CN106795525A - Insect polypeptide and application thereof is killed with broad spectrum of activity - Google Patents

Abstract

Present disclose provides nucleic acid and its variant and fragment that the coding obtained from bacillus thuringiensis bacterial strain has the polypeptide to the insecticidal activity of insect pest (including Lepidoptera and coleopteron).The specific embodiment of the disclosure provides the microorganism and plant of seperated nuclear acid, the Pesticidal combination of the nucleic acid comprising embodiment of the present invention, DNA construct and the conversion of encoding insecticidal proteins.These compositions can be used in the method for pest control particularly plant insect.

Description

Insecticidal polypeptide with broad-spectrum activity and use thereof

References to Sequence Listings Submitted Electronically

A sequence listing created on September 24, 2015 with a size of 59 kilobytes and a file name of "5383WOPCT_SequenceListing.txt" is submitted simultaneously with this specification in computer-readable form. This Sequence Listing is part of this specification and is hereby incorporated by reference in its entirety.

technical field

The present disclosure relates to natural and recombinant nucleic acids obtained from novel Bacillus thuringiensis genes encoding insecticidal polypeptides characterized by insecticidal activity against insect pests. The compositions and methods of the present disclosure utilize the disclosed nucleic acids and the pesticidal polypeptides they encode to control plant pests.

Background technique

Insect pests are a major contributor to crop loss worldwide. For example, armyworm feeding, black cutworm damage, or European corn borer damage can be economically damaging to agricultural producers. Insect pest-related crop losses from European corn borer attack on non-sweet and sweet corn alone cost approximately $1 billion a year in damage and control costs.

Traditionally, the main method of affecting insect pest populations has been the application of broad-spectrum chemical insecticides. However, both consumers and government regulators are increasingly concerned about the environmental hazards associated with the production and use of synthetic chemical pesticides. Because of these concerns, regulators have banned or restricted the use of some of the more hazardous pesticides. Therefore, there is great interest in developing alternative insecticides.

Biological control of insect pests of agricultural interest using microbial agents such as fungi, bacteria, or another insect provides an environmentally friendly and commercially attractive alternative to synthetic chemical insecticides. In general, the use of biopesticides poses a lower risk of contamination and environmental harm, and biopesticides can provide higher target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides tend to be less expensive to produce and thus increase the economic yield of many crops.

Certain species of microorganisms of the genus Bacillus are known to have insecticidal activity against a variety of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera (Hemiptera) and so on. Bacillus thuringiensis (Bt) and Bacillus papilliae are representatives of the most successful biological control agents discovered so far. Strains of B. larvae, B. lentimorbus, B. sphaericus (Harwook ed., (1989), Bacillus (Plenum Press), 306) and strains of B. cereus (WO 96/10083) have also been indicated to be entomopathogenic. Insecticidal activity appeared to be concentrated in parasporal crystal protein inclusion bodies, but insecticidal proteins were also isolated from the vegetative phase of Bacillus. Several genes encoding these pesticidal proteins have been isolated and characterized (see eg US Patents 5,366,892 and 5,840,868).

Microbial insecticides, especially those obtained from Bacillus strains, have played an important role in agriculture as an alternative to chemical pest control. More recently, agricultural scientists have developed crops with enhanced insect resistance by genetically engineering the crops to produce insecticidal proteins from Bacillus bacterium. For example, corn and cotton crops have been genetically engineered to produce insecticidal proteins isolated from strains of Bt (see, e.g., Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998 ) Microbiol Mol Biol Rev. 62(3):775-806). These genetically engineered crops are now widely used in U.S. agriculture, offering farmers an environmentally friendly alternative to traditional insect control methods. Separately, potatoes genetically engineered to contain insecticidal Cry toxins have been sold to US farmers. While these genetically engineered insect-resistant crops have proven to be very commercially successful, they are only resistant to a narrow range of economically important insect pests.

Accordingly, there remains a need for new Bt toxins with a wider range of insecticidal activity against insect pests, eg, toxins active against a wider variety of Lepidoptera. Additionally, there remains a need for biopesticides that are active against a variety of insect pests and for biopesticides with improved insecticidal activity.

Contents of the invention

The present invention provides compositions and methods for affecting insect pests. More specifically, embodiments of the present disclosure relate to methods of affecting insects using nucleotide sequences encoding insecticidal peptides to produce transformed microorganisms and plants expressing the insecticidal polypeptides of the embodiments. In some embodiments, the nucleotide sequence encodes a polypeptide having insecticidal activity against at least one insect belonging to the order Lepidoptera.

Embodiments of the present invention provide nucleic acid molecules encoding polypeptides having insecticidal activity against insect pests and fragments and variants thereof (such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and encoding SEQ ID NO : 2, the polypeptides of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8). The wild-type (eg, native) nucleotide sequence of an embodiment of the invention obtained from Bt encodes an insecticidal peptide. Embodiments of the invention also provide fragments and variants of the disclosed nucleotide sequences that encode biologically active (eg, insecticidal) polypeptides.

Embodiments of the invention also provide isolated pesticidal (eg, insecticidal) polypeptides encoded by native or modified (eg, mutagenized or manipulated) nucleic acids of embodiments of the invention. As specific examples, the pesticidal proteins of the embodiments of the present invention include fragments of full-length proteins and polypeptides produced from mutagenized nucleic acids designed to introduce specific amino acid sequences into the in peptides. In certain embodiments, the polypeptides have enhanced pesticidal activity relative to the pesticidal activity of the native polypeptide from which they are derived.

Nucleic acids according to embodiments of the invention can also be used to produce transgenic (e.g., transformed) monocotyledonous or dicotyledonous plants characterized in that their genome comprises at least one stably incorporated nucleotide construct, the nuclear The nucleotide construct comprises the coding sequence of an embodiment of the invention operably linked to a promoter driving expression of the encoded pesticidal polypeptide. Accordingly, the present invention also provides transformed plant cells, plant tissues, plants and seeds thereof.

In a specific embodiment, a nucleic acid that has been optimized for increased expression in a host plant can be used to generate transformed plants. For example, one of the pesticidal polypeptides of embodiments of the invention can be back-translated to produce a nucleic acid comprising codons optimized for expression in a particular host, such as a crop such as a corn (maize) crop. Expression of the coding sequence by such transformed plants (eg, dicots or monocots) will result in the production of the pesticidal polypeptide and confer increased insect resistance to the plant. Some embodiments provide transgenic plants expressing pesticidal polypeptides that can be used in methods for affecting various insect pests.

Embodiments of the present invention also include pesticidal or insecticidal compositions comprising the insecticidal polypeptides of embodiments of the present invention, and may optionally contain additional insecticidal peptides. Embodiments of the present invention contemplate the application of such compositions to the environment of insect pests to affect insect pests.

Description of drawings

Figures 1A-1D show the sequence alignment of Cry1JPD166 polypeptide (SEQ ID NO: 2), Cry1JP578V polypeptide (SEQ ID NO: 4), Cry1JPS1 polypeptide (SEQ ID NO: 6) and Cry1JPS1P578V (SEQ ID NO: 8). Amino acid differences are shaded. The amino acid substitution of proline to valine at position 578 is indicated by a "◆" above the amino acid position. Amino acid substitutions at positions 115, 594 and 794 resulting from optimization of soybean expression genes encoding Cry1JPS1 (SEQ ID NO: 6) and Cry1JPS1P578V (SEQ ID NO: 8) are indicated by "•" above the amino acid positions.

detailed description

Embodiments of the present disclosure relate to compositions and methods for affecting insect pests, especially plant pests. More specifically, isolated nucleic acids of embodiments of the invention, and fragments and variants thereof, comprise a nucleotide sequence encoding a pesticidal polypeptide (eg, protein). The disclosed pesticidal proteins have biological activity (eg, pesticidal activity) against insect pests, such as, but not limited to, insect pests of the order Lepidoptera.

Compositions of embodiments of the present invention comprise isolated nucleic acids encoding pesticidal polypeptides and fragments and variants thereof, expression cassettes comprising nucleotide sequences of embodiments of the present invention, isolated pesticidal proteins, and pesticidal compositions. Some embodiments provide modified insecticidal polypeptides having improved insecticidal activity against Lepidopteran insects relative to the insecticidal activity of a corresponding wild-type protein. Embodiments of the invention also provide plants and microorganisms transformed with these novel nucleic acids, and methods involving the use of such nucleic acids, pesticidal compositions, transformed organisms, and products thereof for affecting insect pests.

The nucleic acids and nucleotide sequences of the embodiments of the invention can be used to transform any organism to produce the encoded pesticidal protein. The present invention provides methods involving the use of such transformed organisms to affect or control plant pests. The nucleic acids and nucleotide sequences of embodiments of the present invention can also be used to transform organelles such as chloroplasts (McBride et al., (1995) Biotechnology 13:362-365; and Kota et al., (1999) Proc.Natl.Acad.Sci.USA 96:1840-1845).

Embodiments of the invention also relate to the identification of fragments and variants of native coding sequences encoding biologically active pesticidal proteins. The nucleotide sequences of the embodiments of the present invention are used directly in methods for affecting pests, especially insect pests such as Lepidopteran pests. Accordingly, embodiments of the present invention provide new methods of affecting insect pests that do not rely on the use of traditional synthetic chemical insecticides. Embodiments of the present invention relate to the discovery of natural biodegradable pesticides and the genes encoding them.

Embodiments of the invention also provide fragments and variants of native coding sequences that also encode biologically active (eg, pesticidal) polypeptides. Nucleic acids of the embodiments of the present invention encompass nucleic acids or nucleotide sequences that have been optimized for expression by cells of a particular organism, e.g., have been back-translated using plant-preferred codons based on the amino acid sequence of a polypeptide with enhanced pesticidal activity ( That is, reverse translation) nucleic acid sequence. Embodiments of the invention also provide mutations that confer improved or altered properties on polypeptides of embodiments of the invention. See, eg, US Patent 7,462,760.

In the following description, various terms are used in a broad manner. The following definitions are provided to facilitate the understanding of embodiments of the present invention.

Units, prefixes and symbols may be expressed in their International System of Units (SI) accepted form. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. Numerical ranges include the numbers defining the range. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined above are more fully defined by reference to the specification as a whole.

As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and unless otherwise limited, encompasses known nucleic acids having the essential properties of natural nucleotides in Analogs (eg, peptide nucleic acids): which hybridize to single-stranded nucleic acids in a manner similar to natural nucleotides.

As used herein, the term "encoding" or "encoded" when used in the context of a designated nucleic acid means that the nucleic acid contains the information necessary to direct the translation of a nucleotide sequence into a designated protein. The information by which a protein is encoded is determined through the use of codons. A nucleic acid encoding a protein may contain non-translated sequences (eg, introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (eg, as in cDNA).

As used herein, "full-length sequence" with respect to a specified polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having native (non-synthetic) endogenous sequences. A full-length polynucleotide encodes a full-length catalytically active form of a given protein.

As used herein, the term "antisense" when used in the context of the orientation of a nucleotide sequence refers to a double stranded polynucleotide sequence operably linked to a promoter in an orientation in which the antisense strand is transcribed. The antisense strand is sufficiently complementary to the endogenous transcript that translation of the endogenous transcript is often inhibited. Thus, where the term "antisense" is used in the context of a particular nucleotide sequence, the term refers to the complementary strand of the reference transcript.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of the corresponding natural amino acid, and to natural amino acid polymers.

The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to amino acids incorporated into proteins, polypeptides or peptides (collectively "proteins"). The amino acid may be a natural amino acid and, unless otherwise limited, encompasses known analogs of natural amino acids that can function in a similar manner to natural amino acids.

Polypeptides of embodiments of the invention can be produced from the nucleic acids disclosed herein, or by using standard molecular biology techniques. For example, proteins of embodiments of the invention can be produced by expressing recombinant nucleic acids of embodiments of the invention in appropriate host cells, or alternatively, by a combination of ex vivo procedures.

As used herein, the terms "isolated" and "purified" are used interchangeably to refer to a nucleic acid or polypeptide, or biologically active portion thereof, that is substantially or essentially free of the components normally found in its natural environment with which it is associated. or interacting components. Thus, an isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when synthesized chemically.

An "isolated" nucleic acid is generally free of sequences (eg, protein coding sequences) that naturally flank the nucleic acid (ie, sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, an isolated nucleic acid may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of a nucleotide sequence that is present in the cell from which the nucleic acid is derived. The nucleic acid is naturally flanked in genomic DNA.

As used herein, the term "isolated" or "purified" when used to refer to a polypeptide of an embodiment of the present invention means that the isolated protein is substantially free of cellular material and includes cells having less than about 30%, 20%, Protein preparations with 10% or 5% (by dry weight) of contaminating protein. When the proteins of embodiments of the invention, or biologically active portions thereof, are produced recombinantly, the culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-target proteins Chemicals.

A "recombinant" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) in a recombinant bacterial or plant host cell. In some embodiments, an "isolated" or "recombinant" nucleic acid is free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., at the 5' and 3' ends of the nucleic acid). sequence) (preferably protein coding sequence). For the purposes of this disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules excludes isolated chromosomes.

As used herein, "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" refers to a nucleic acid molecule that has one or more alterations in the nucleic acid sequence as compared to a native or genomic nucleic acid sequence. In some embodiments, changes in native or genomic nucleic acid molecules include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; or genomic sequence, in order to introduce at least one amino acid substitution, insertion, deletion, and/or addition in the nucleic acid sequence; the removal of one or more introns associated with the genomic nucleic acid sequence; one or more Insertion of a heterologous intron; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; 5 associated with the genomic nucleic acid sequence deletion of ' and/or 3' untranslated regions; insertion of heterologous 5' and/or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

Throughout this specification, the word "comprise" and its grammatical variants should be understood as implying the inclusion of stated elements, integers or steps or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps or Group of elements, groups of integers or groups of steps.

As used herein, the term "affecting insect pests" means causing changes in insect feeding, growth and/or behavior at any stage of insect development, including but not limited to: killing insects; delaying growth; hindering reproductive capacity; antifeedant activity Wait.

As used herein, the terms "pesticidal activity" and "insecticidal activity" are used synonymously to refer to the ability of an organism or substance (such as, for example, a protein) to pass through pests after feeding and exposure for an appropriate length of time, pest mortality, Activity measured by pest weight loss, pest repellency, and other behavioral and physical changes in the pest (but not limited to). Thus, an organism or substance having pesticidal activity adversely affects at least one measurable parameter of pest fitness. For example, a "pesticidal protein" is a protein that exhibits pesticidal activity by itself or in combination with other proteins.

As used herein, the term "pesticidally effective amount" means an amount of a substance or organism that is pesticidally active when present in the environment of the pest. For each substance or organism, the pesticidally effective amount is determined empirically for each pest affected in the given environment. Similarly, "insecticidally effective amount" may be used to mean "insecticidally effective amount" when the pest is an insect pest.

As used herein, the term "recombinantly engineered" or "engineered" refers to the introduction (such as engineering transformation) changes.

As used herein, the term "mutated nucleotide sequence" or "mutation" or "mutated nucleotide sequence" means a nucleotide sequence that has been mutagenized or altered to contain one or more of the Nucleotide residues (eg, base pairs) that are absent in the corresponding wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions or substitutions or substitutions of nucleic acid residues. When mutations are made by adding, removing or substituting amino acids at proteolytic sites, such additions, removals or substitutions may be within or near the proteolytic site motif, as long as the purpose of the mutation is achieved (ie, as long as proteolysis at that site is altered).

A mutant nucleotide sequence may encode a mutant insecticidal toxin exhibiting improved or reduced insecticidal activity, or an amino acid sequence that confers improved or reduced insecticidal activity on a polypeptide containing it. As used herein, the term "mutant" or "mutation" in the context of a protein, polypeptide or amino acid sequence refers to a sequence that has been mutagenized or altered to contain one or more mutations present in the corresponding wild-type sequence. Amino acid residues that do not exist. Such mutagenesis or alteration consists of one or more additions, deletions or substitutions or substitutions of amino acid residues. A mutant polypeptide exhibits improved or reduced insecticidal activity, or represents an amino acid sequence that confers improved insecticidal activity on a polypeptide containing it. Thus, the term "mutant" or "mutation" refers to either or both of the mutated nucleotide sequence and the encoded amino acid. Mutants may be used alone or in any compatible combination with other mutants of embodiments of the invention or with other mutants. "Mutant polypeptides" may conversely exhibit reduced insecticidal activity. Where more than one mutation is added to a particular nucleic acid or protein, the mutations can be added simultaneously or sequentially; if added sequentially, the mutations can be added in any suitable order.

As used herein, the term "improved insecticidal activity" or "improved insecticidal activity" refers to that the insecticidal polypeptide of the present invention has enhanced insecticidal activity relative to its corresponding wild-type protein, and/or the The insecticidal polypeptide is effective against a wider range of insects, and/or the insecticidal polypeptide is specific for insects that are less susceptible to the toxicity of the wild-type protein. The discovery of improved or enhanced insecticidal activity requires demonstrating an increase in the insecticidal activity of the insect target by at least 10%, or by at least 20%, relative to the insecticidal activity of the wild-type insecticidal polypeptide measured against the same insect target , 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, or 300% or more.

For example, improved insecticidal or insecticidal activity is provided if the range of insects affected by the polypeptide is wider or narrower relative to the range of insects affected by the wild-type Bt toxin. A wider range of effect may be desirable where versatility is required, whereas a narrower range of effect may be desirable where, for example, beneficial insects may instead be affected by the use or presence of the toxin . Although embodiments of the invention are not bound by any particular mechanism of action, improved insecticidal activity may also be provided by alteration of one or more properties of the polypeptide; for example, the stability or longevity of the polypeptide in the insect gut may be compared to The stability or longevity of the corresponding wild-type protein is improved.

As used herein, the term "toxin" refers to a polypeptide exhibiting pesticidal activity or insecticidal activity or improved pesticidal activity or improved insecticidal activity. "Bt" or "Bacillus thuringiensis" toxins are intended to include the broader class of Cry toxins present in various strains of Bacillus thuringiensis, including toxins such as, for example, Cry1s, Cry2s, or Cry3s.

The term "proteolytic site" or "cleavage site" refers to an amino acid sequence that confers sensitivity to a class of protease or a specific protease such that a polypeptide containing the amino acid sequence is cleaved by the class of protease or the specific protease Digestion. A proteolytic site is said to be "sensitive" to proteases that recognize the site. It is recognized in the art that the efficiency of digestion can vary and that a decrease in the efficiency of digestion can lead to increased stability or longevity of the polypeptide in the insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at the site may vary for various proteases. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.

Studies have confirmed that intestinal proteases of Lepidoptera insects include trypsin, chymotrypsin and elastase. See eg Lenz et al., (1991) Arch. Insect Biochem. Physiol. 16:201-212; and Hedegus et al., (2003) Arch. Insect Biochem. Physiol. 53:30-47. For example, approximately 18 different trypsinases are found in the midgut of Helicoverpa armigera larvae (see Gatehouse et al. (1997) Insect Biochem. Mol. Biol. 27:929-944). The preferred proteolytic substrate sites of these proteases have been investigated. See, eg, Peterson et al., (1995) Insect Biochem. Mol. Biol. 25:765-774.

Attempts have been made to understand the mechanism of action of Bt toxins and to engineer toxins with improved properties. Insect intestinal proteases have been shown to affect the effects of Bt Cry proteins on insects. Some proteases activate Cry proteins by processing them from a "protoxin" form to a toxic form or "toxin". See Oppert (1999) Arch. Insect Biochem. Phys. 42:1-12; and Carroll et al., (1997) J. Invertebrate Pathology 70:41-49. Such activation of the toxin can include removal of the N-terminal and C-terminal peptides from the protein, and can also include internal cleavage of the protein. Other proteases can degrade Cry proteins. See Oppert (supra).

Comparison of the amino acid sequences of Cry toxins with different specificities revealed five highly conserved sequence blocks. Structurally, the toxin contains three distinct domains, which are, from N-terminus to C-terminus: a cluster of seven alpha helices (termed "domain 1") involved in pore formation, three antiparallel domains involved in cell binding The beta sheet (referred to as "domain 2") and the beta sandwich (referred to as "domain 3"). The location and nature of these domains are known to those skilled in the art. See eg Li et al., (1991) Nature, 305:815-821 and Morse et al., (2001) Structure, 9:409-417. When referring to a particular domain, such as domain 1, it is understood that the exact end point of the domain relative to the particular sequence is not critical, so long as the sequence or portion thereof includes a protein that provides at least some of the function attributable to the particular domain. sequence. Thus, for example, when referring to "domain 1" it is meant that the particular sequence comprises a cluster of seven alpha helices, but the exact terminus of the sequence used or referred to with respect to the cluster is not critical. Those skilled in the art are familiar with the determination of such endpoints and the assessment of such functions.

To better characterize and improve Bt toxins, various strains of bacterial Bt have been studied. Crystal preparations prepared from cultures of Bt strains were found to have insecticidal activity against various Lepidopteran pests (see, eg, Experimental Example 1). Efforts have been made to identify nucleotide sequences encoding crystal proteins from selected bacterial strains, and wild-type (i.e. native) nucleic acids of embodiments of the present invention have been isolated from these bacterial strains, cloned into expression vectors and transformed into in Escherichia coli. It is recognized that, depending on the properties of a given preparation, trypsin pretreatment is sometimes required to activate pesticidal proteins in order to exhibit pesticidal activity. Thus, it will be appreciated that some pesticidal proteins require protease digestion (eg, by trypsin, chymotrypsin, etc.) for activation, while other proteins are biologically active (eg, pesticidal) without activation.

Such molecules can be altered by means such as described in US Patent No. 7,462,760. In addition, nucleic acid sequences can be engineered to encode polypeptides that contain additional mutations that confer improved or altered pesticidal activity relative to the pesticidal activity of the native polypeptide. The nucleotide sequence of such an engineered nucleic acid contains mutations that are not present in the wild-type sequence.

Mutant polypeptides according to embodiments of the present invention are usually prepared by a process comprising the steps of: obtaining a nucleic acid sequence encoding a Cry family polypeptide; analyzing the structure of the polypeptide based on consideration of the proposed target domain's function in the toxin's mode of action to identify A specific "target" site for mutagenesis of a corresponding gene sequence; the introduction of one or more mutations into the nucleic acid sequence to produce the desired change in one or more amino acid residues of the encoded polypeptide sequence and determining the pesticidal activity of the polypeptide produced.

Many Bt insecticidal toxins are related to varying degrees due to their amino acid sequence and tertiary structure similarities, and means for obtaining crystal structures of Bt toxins are well known. Exemplary high resolution crystal structure solutions for both Cry3A and Cry3B polypeptides are available in the literature. The solved structure of the Cry3A gene (Li et al., (1991) Nature 353:815-821) has provided insight into the relationship between structure and function of the toxin. Considering published structural analyzes of Bt toxins in conjunction with reported functions associated with specific structures, motifs, etc., specific regions of the toxin were found to be associated with specific functions and discrete steps of the protein's mode of action. For example, many toxins isolated from Bt are generally described as comprising three domains: a seven-helix bundle involved in pore formation, a three-sheet domain involved in receptor binding, and a β-sandwich motif (Li et al., (1991) Nature 305:815-821).

As reported in US Patent Nos. 7,105,332 and 7,462,760, the toxicity of this toxin can be improved by targeting the region between alpha helices 3 and 4 of domain 1 of the Cry protein. The theory is premised on a wealth of knowledge about insecticidal toxins, including: 1) Alpha helices 4 and 5 of domain 1 of the Cry3A toxin have been reported to insert into the lipid bilayer of cells lining the midgut of susceptible insects (Gazit et al., (1998) Proc.Natl.Acad.Sci.USA 95:12289-12294); 2) the inventors knew the position of the trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) The wild type protein was observed to be more active against certain insects after in vitro activation by trypsin or chymotrypsin treatment; and 4) Digestion of the toxin from the 3' end has been reported to result in reduced toxicity to insects.

A series of mutations can be generated and placed in multiple background sequences to generate novel polypeptides with enhanced or altered pesticidal activity. See, eg, US Patent 7,462,760. These mutants include, but are not limited to: adding at least one more protease-sensitive site (such as a trypsin cleavage site) in the region between helices 3 and 4 of domain 1; The sensitive site is replaced with a different protease sensitive site; multiple protease sensitive sites are added in a specific position; amino acid residues are added near the protease sensitive site to change the folding of the polypeptide to enhance the polypeptide in the protease sensitive site and adding mutations to protect the polypeptide from degradative digestion that would reduce toxicity (eg, making a series of mutations in which the wild-type amino acid is substituted with valine to protect the polypeptide from digestion). The individual mutations may be used alone or in any combination to provide polypeptides according to embodiments of the invention.

Homologous sequences were identified by similarity searches in the non-redundant database (nr) of the National Center for Biotechnology Information (NCBI) using BLAST and PSI-BLAST. Homologous proteins consist of the Cry toxin mainly from Bacillus thuringiensis.

Mutations as additional or alternative protease sensitive sites may be sensitive to several classes of proteases, such as serine proteases (which include trypsin and chymotrypsin) or enzymes such as elastase. Accordingly, mutations to additional or alternative protease sensitive sites can be designed such that the site is readily recognized and/or cleaved by a range of proteases, such as mammalian proteases or insect proteases. Protease sensitive sites can also be designed to be cleaved by specific classes of enzymes or specific enzymes known to be produced in certain organisms, such as, for example, chymotrypsin produced by Heliothis zea (Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16:201-212). Mutations may also confer resistance to proteolytic digestion, for example resistance to chymotrypsin digestion at the C-terminus of the peptide.

The presence of additional and/or alternative protease-sensitive sites in the amino acid sequence of the polypeptide encoded by the nucleic acid of the present invention can improve the insecticidal activity and/or specificity of the encoded polypeptide. Accordingly, the nucleotide sequences of the embodiments of the invention can be recombinantly engineered or manipulated to produce polypeptides with improved or altered insecticidal activity and/or specificity compared to unmodified wild-type toxins. In addition, the mutations disclosed herein can be placed into or used in conjunction with other nucleotide sequences to provide improved properties. For example, a protease-sensitive site that is easily cleaved by insect chymotrypsin, such as that present in shawl armyworm or corn earworm (Hegedus et al., (2003) Arch. Insect Biochem. Physiol. 53 : 30-47; and Lenz et al., (1991) Arch. Insect Biochem. Physiol. 16: 201-212) into the Cry background sequence to provide improved toxicity to that sequence. Thus, embodiments of the present invention provide toxic polypeptides with improved properties.

For example, the mutagenized Cry nucleotide sequence can constitute an additional mutant containing an additional codon that introduces a second trypsin-sensitive amino acid sequence (outside the native trypsin site) into the encoded in peptides. Alternative addition mutants of embodiments of the invention comprise additional codons designed to introduce at least one additional, different protease site into the polypeptide, such as immediately 5' or 3' to the native trypsin site Chymotrypsin sensitive site. Alternatively, substitution mutants can be produced in which at least one codon encoding a native protease sensitive site of the nucleic acid is disrupted and alternative codons are introduced into the nucleic acid sequence to provide a different (e.g. substituted) protease sensitive site. Substitution mutants can also be added to the Cry sequence in which the native trypsin cleavage site present in the encoded polypeptide is disrupted and a chymotrypsin or elastase cleavage site is introduced in its place.

It is recognized that any nucleotide sequence encoding an amino acid sequence that is or is a putative proteolytic site (for example a sequence such as RR or LKM) can be used and used to introduce any of these cleavage sites The exact identity of codons incorporated into the variant polypeptide may vary according to usage (ie, expression) in a particular plant species. It is also recognized that any of the disclosed mutations may be introduced into any polynucleotide sequence of an embodiment of the invention comprising an amino acid residue that provides the native trypsin cleavage site targeted for modification. a. Accordingly, variants of the full-length toxin or fragments thereof may be modified to contain additional or alternative cleavage sites, and such embodiments are intended to be encompassed within the scope of the embodiments disclosed herein.

Those skilled in the art will recognize that any useful mutation may be added to the sequences of the embodiments of the invention so long as the encoded polypeptide retains pesticidal activity. Thus, the sequence may also be mutated such that the encoded polypeptide is resistant to proteolytic digestion by chymotrypsin. More than one recognition site can be added at a particular position in any combination, and multiple recognition sites can be added to or removed from the toxin. Thus, additional mutations may comprise three, four or more recognition sites. It will be appreciated that multiple mutations can be engineered in any suitable polynucleotide sequence; thus, either the full-length sequence or fragments thereof can be modified to contain additional or alternative cleavage sites as well as to be resistant to protein Hydrolytic digestion. Thus, embodiments of the present invention provide Cry toxins containing mutations that improve pesticidal activity, as well as improved compositions and methods for affecting pests using other Bt toxins.

Mutations can protect the polypeptide from degradation by proteases, for example, by removing putative proteolytic sites (eg, putative serine protease sites and elastase recognition sites) from various regions. Some or all of these putative sites can be removed or altered such that proteolysis is reduced at the original site location. Changes in proteolysis can be assessed by comparing the mutant polypeptide to wild-type toxin, or by comparing individual mutant toxins that differ in amino acid sequence. Putative proteolytic sites and proteolytic sites include, but are not limited to, the following sequences: RR, which is a trypsin cleavage site; LKM, which is a chymotrypsin site; and a trypsin site. These positions can be altered by adding or deleting any number and kind of amino acid residues as long as the pesticidal activity of the polypeptide is improved. Thus, a polypeptide encoded by a nucleotide sequence comprising a mutation will comprise, relative to the native or background sequence, at least one amino acid change or addition, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47 ,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270 or 280 or More amino acid changes or additions. The pesticidal activity of a polypeptide can also be improved by truncation of the native or full-length sequence, as is known in the art.

Compositions of embodiments of the present invention include nucleic acids encoding pesticidal polypeptides and fragments and variants thereof. Specifically, the embodiments of the present invention provide an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or encoding the amino acid sequence Nucleotide sequences of the aforementioned amino acid sequences, such as the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and fragments and variants thereof.

Specifically, embodiments of the present invention provide an isolated nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8 or a nucleotide sequence encoding the amino acid sequence, such as SEQ ID NO: 3 or SEQ ID Nucleotide sequences shown in NO: 7, and fragments and variants thereof.

Also of interest are optimized nucleotide sequences encoding pesticidal proteins of embodiments of the present invention. As used herein, the phrase "optimized nucleotide sequence" refers to a nucleic acid that is optimized for expression in a particular organism, such as a plant. Optimized nucleotide sequences can be prepared for any organism of interest using methods known in the art. See, eg, US Patent 7,462,760, which describes optimized nucleotide sequences encoding the disclosed pesticidal proteins. In this example, the nucleotide sequence was prepared by back-translating the amino acid sequence of the protein and altering the nucleotide sequence to contain maize preferred codons while still encoding the same amino acid sequence. Murray et al. (1989) Nucleic Acids Res. 17:477-498 describe this procedure in more detail. The optimized nucleotide sequence can be used to increase the expression of the insecticidal protein in plants, such as monocotyledonous plants of Gramineae (Gramineae, Poaceae), such as maize or maize plants.

More specifically, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, as well as fragments and variants thereof, are provided.

In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8, and fragments and variants thereof. In some embodiments, the polypeptide fragment comprises Cry domain I, domain II and domain III of SEQ ID NO:4 or SEQ ID NO:8.

In particular embodiments, the pesticidal proteins of embodiments of the invention provide full-length insecticidal polypeptides, fragments of full-length insecticidal polypeptides, and mutagenized proteins from those designed to introduce specific amino acid sequences into polypeptides of embodiments of the invention. Variant polypeptides produced from nucleic acids. In certain embodiments, the amino acid sequence introduced into the polypeptide comprises a sequence that provides a cleavage site for an enzyme, such as a protease.

It is known in the art that the insecticidal activity of Bt toxins is usually activated by cleavage of the peptide by various proteases in the gut of insects. Since peptides may not always be cleaved with full efficiency in the insect gut, fragments of the full-length toxin may have enhanced insecticidal activity compared to the full-length toxin itself. Accordingly, some of the polypeptides of the present embodiments include fragments of full-length insecticidal polypeptides, and some of the polypeptide fragments, variants and mutations have enhanced insecticidal activity compared to the native insecticidal polypeptides from which they are derived , especially if the natural insecticidal polypeptide is not activated in vitro with proteases prior to screening for activity. Accordingly, this application encompasses truncated versions or fragments of said sequences.

Mutations can be placed into any background sequence, including such truncated polypeptides, so long as the polypeptide retains pesticidal activity. One of skill in the art can readily compare the pesticidal activity of two or more proteins using assays known in the art or described elsewhere herein. It is understood that the polypeptides of the embodiments of the invention can be produced by expressing the nucleic acids disclosed herein, or by using standard molecular biology techniques.

It is recognized that pesticidal proteins can be oligomeric and vary in molecular weight, number of residues, constituent peptides, activity against specific pests, and other characteristics. However, by the methods presented herein, proteins active against a wide variety of pests can be isolated and characterized. The insecticidal proteins of embodiments of the present invention can be used in combination with other Bt toxins or other insecticidal proteins to increase the range of insect targets. Furthermore, the insecticidal proteins of embodiments of the present invention have particular utility for preventing and/or managing insect resistance when used in combination with other Bt toxins or other insecticidal principles with unique properties. Other insecticides include protease inhibitors (both serine and cysteine types), alpha-amylases and peroxidases.

Fragments and variants of nucleotide and amino acid sequences and the polypeptides they encode are also encompassed by embodiments of the invention. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of the embodiments of the present invention. Fragments of the nucleotide sequence may encode protein fragments that retain the biological activity of the native or corresponding full-length protein and thus have insecticidal activity. Accordingly, it is recognized that some of the polynucleotide and amino acid sequences of the embodiments of the present invention may properly be referred to as both fragments and mutants.

It should be understood that the term "fragment" when used to refer to a nucleic acid sequence of an embodiment of the present invention also encompasses sequences that can be used as hybridization probes. Such nucleotide sequences generally do not encode fragmented proteins that retain biological activity. Accordingly, fragments of a nucleotide sequence can be at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides in length and up to a full-length nucleotide sequence encoding a protein of an embodiment of the invention.

Fragments of the nucleotide sequences of the embodiments of the invention encoding the biologically active portion of the pesticidal protein of the embodiments of the invention will encode at least 15, 25, 30, 50, 100, 200, 250 or 300 consecutive amino acids or up to the present The total number of amino acids present in the pesticidal polypeptide of an embodiment of the invention (eg 1167 amino acids for SEQ ID NO: 2). Accordingly, it should be understood that embodiments of the present invention also encompass polypeptides that are fragments of the exemplary pesticidal proteins of embodiments of the present invention and are at least 15, 25, 30, 50, 100, 200, 250, or 300 in length Contiguous amino acids or up to the total number of amino acids present in a pesticidal polypeptide of an embodiment of the invention (eg 1167 amino acids for SEQ ID NO: 2). Fragments of the nucleotide sequences of the embodiments of the invention that are useful as hybridization probes or PCR primers generally need not encode biologically active portions of pesticidal proteins. Thus, a fragment of a nucleic acid of an embodiment of the invention may encode a biologically active portion of a pesticidal protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed herein. A biologically active portion of a pesticidal protein can be prepared by isolating a portion of one of the nucleotide sequences of an embodiment of the invention, expressing the coding portion of the pesticidal protein (e.g., by recombinant expression in vitro), and evaluating the coding portion of the pesticidal protein. part of the activity.

Nucleic acids that are fragments of nucleotide sequences according to embodiments of the invention comprising at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 850 , 900 or 950 nucleotides or up to the number of nucleotides present in the nucleotide sequences disclosed herein (eg 3501 nucleotides for SEQ ID NO: 1). Particular embodiments contemplate fragments derived from (eg, produced from) the first nucleic acid of embodiments of the invention, wherein the fragment encodes a truncated toxin having pesticidal activity. Truncated polypeptides encoded by polynucleotide fragments of embodiments of the invention have pesticidal activity that is comparable or improved relative to the activity of the corresponding full-length polypeptide encoded by the first nucleic acid from which the fragment is derived. It is envisioned that such nucleic acid fragments of embodiments of the invention may be truncated at the 3' end of the native or corresponding full-length coding sequence. Nucleic acid fragments may also be truncated at both the 5' and 3' ends of the native or corresponding full-length coding sequence.

The term "variant" is used herein to refer to substantially similar sequences. With respect to nucleotide sequences, conservative variants include those that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the pesticidal polypeptides of the embodiments of the present invention. Those of ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, there are a large number of encoding nucleotide sequences of the present disclosure.

In some embodiments, a nucleic acid molecule encoding a polypeptide is a non-genomic nucleic acid sequence. As used herein, "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule having one or more alterations in the nucleic acid sequence as compared to a native or genomic nucleic acid sequence. In some embodiments, changes in native or genomic nucleic acid molecules include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; or genomic sequence, in order to introduce at least one amino acid substitution, insertion, deletion, and/or addition in the nucleic acid sequence; the removal of one or more introns associated with the genomic nucleic acid sequence; one or more Insertion of a heterologous intron; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; 5 associated with the genomic nucleic acid sequence deletion of ' and/or 3' untranslated regions; insertion of heterologous 5' and/or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is cDNA. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

Where appropriate, nucleic acids may be optimized for increased expression in the host organism. Thus, if the host organism is a plant, synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. For a discussion of host-preferred codon usage, see, eg, Campbell and Gowri, (1990) Plant Physiol. 92:1-11. For example, although the nucleic acid sequences of the embodiments of the invention are expressible in both monocot and dicot species, the sequences can be modified to account for the specific codon preferences and GC content preferences of either monocots or dicots. because these preferences have been shown to be different (Murray et al., (1989) Nucleic Acids Res. 17:477-498). Thus, maize preferred codons for specific amino acids can be derived from known gene sequences from maize. The maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra. Methods for synthesizing plant-preferred genes are readily available in the art. See, eg, US Patents 5,380,831 and 5,436,391 and Murray et al., (1989) Nucleic Acids Res. 17:477-498 and Liu H et al., Mol Bio Rep 37:677-684, 2010, all incorporated herein by reference. A maize codon usage table can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi? species=4577 (accessible with www prefix).

Soybean (Glycine max) codon usage table is shown in Table 3 and can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi? species=3847&aa=1&style=N (accessible with www prefix).

The skilled artisan will also appreciate that changes may be introduced by mutation of the nucleic acid sequence, resulting in a change in the amino acid sequence of the encoded polypeptide without altering the biological activity of the protein. Thus, variant nucleic acid molecules can be formed by introducing one or more nucleotide substitutions, additions and/or deletions into the corresponding nucleic acid sequences disclosed herein such that one or more amino acid substitutions, additions or deletions are introduced into the encoded in the protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present invention.

Natural allelic variants such as these can be identified using well known molecular biology techniques such as, for example, the polymerase chain reaction (PCR) and hybridization techniques described herein.

In some embodiments, the polynucleotide encoding the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 8 is a non-genomic nucleic acid sequence.

Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, eg, by using site-directed mutagenesis, but still encode pesticidal proteins (eg, mutant toxins) of embodiments of the invention. Typically, variants of a particular nucleotide sequence of the present embodiments will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity by sequence alignment programs described elsewhere herein using default parameters Determination. Variants of the nucleotide sequences of the embodiments of the present invention may differ from the sequence by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3 , 2 or even 1 nucleotide.

Variants of specific nucleotide sequences (i.e., exemplary nucleotide sequences) of the embodiments of the present invention can also be determined by comparing the polypeptide encoded by the variant nucleotide sequence with the polypeptide encoded by the reference nucleotide sequence. The percent sequence identity was evaluated. Thus, for example, an isolated nucleic acid encoding a polypeptide having a given percent sequence identity to a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 is disclosed. The percent sequence identity between any two polypeptides can be calculated using the default parameters using a sequence alignment program described elsewhere herein. Where any given pair of polynucleotides of the embodiments of the invention is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the sequence identity between the two encoded polypeptides The percentage is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, usually at least about 75%, 80%, 85%, at least about 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, or at least about 98%, 99% or greater sequence identity.

As used herein, the term "variant protein" encompasses a polypeptide derived from a native protein by deletion (so-called truncation) or addition of one or more amino acids at the N- and/or C-terminus of the native protein; Deletion or addition of one or more amino acids at one or more positions in a protein; or substitution of one or more amino acids at one or more positions in the native protein. Thus, the term "variant protein" encompasses biologically active fragments of the native protein that contain a sufficient number of contiguous amino acid residues to retain the biological activity of the native protein, ie possess pesticidal activity. This pesticidal activity may be different or improved relative to the native protein, or it may be unchanged so long as the pesticidal activity is maintained.

Variant proteins encompassed by embodiments of the present invention are biologically active, ie they continue to possess the desired biological activity of the native protein, ie the pesticidal activity described herein. Such variants may result, for example, from genetic polymorphisms or from human manipulation. Biologically active variants of native pesticidal proteins of embodiments of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88% of the amino acid sequence of the native protein , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, which is determined by using the sequence comparison described elsewhere herein The default parameters were determined for the program. Biologically active variants of proteins according to embodiments of the invention may differ from the protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, or 3 , 2 or even 1 amino acid residue.

In some embodiments, the insecticidal polypeptide has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.

In some embodiments, polypeptides have improved physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical properties of a protein. As used herein, "physical property of interest" and "property of interest" are used interchangeably to refer to the physical property of the protein being studied and/or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on a protein surface, net hydrophobicity and hydrophobic residue distribution on a protein surface, surface charge density, surface hydrophobicity density, total number of surface ionizable groups, surface Tension, protein size and its distribution in solution, melting temperature, heat capacity and second virial coefficient. Examples of physical properties also include, but are not limited to, solubility, foldability, stability, and digestibility. In some embodiments, the polypeptide enhances the digestibility of the proteolytic fragment in the insect gut. In some embodiments, the polypeptide has enhanced stability in the insect gut. Models of digestion by simulated gastric juice are known to those skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50:83-88, 1996; Astwood, J.D. et al., Nature Biotechnology 14:1269-1273, 1996; Fu TJ et al., J. Agric Food Chem. 50:7154-7160, 2002).

Embodiments of the invention also encompass microorganisms transformed with at least one nucleic acid of embodiments of the invention, transformed with an expression cassette comprising the nucleic acid, or transformed with a vector comprising the expression cassette. In some embodiments, the microorganism is a plant-propagating microorganism. One embodiment of the present disclosure relates to encapsulated pesticidal proteins comprising transformed microorganisms capable of expressing at least one pesticidal protein of an embodiment of the invention.

Embodiments of the present invention provide pesticidal compositions comprising transformed microorganisms of embodiments of the present invention. In such embodiments, the transformed microorganism is typically present in a pesticidal composition in a pesticidally effective amount together with a suitable carrier. Embodiments of the present invention also encompass pesticidal compositions comprising an isolated protein of an embodiment of the invention alone or in combination with an isolated protein of an embodiment of the invention in an insecticidally effective amount and a suitable carrier. A combination of transformed microorganisms and/or encapsulated pesticidal proteins according to embodiments of the present invention.

Embodiments of the present invention also provide methods of increasing the range of insect targets by using pesticidal proteins of embodiments of the present invention in combination with at least one other or "second" pesticidal protein. Any pesticidal protein known in the art may be employed in the methods of embodiments of the present invention. Such pesticidal proteins include, but are not limited to, Bt toxins, protease inhibitors, alpha-amylases and peroxidases.

Embodiments of the invention also encompass transformed or transgenic plants comprising at least one nucleotide sequence of an embodiment of the invention. In some embodiments, the plant is stably transformed with a nucleotide construct comprising at least one nucleoside of an embodiment of the invention operably linked to a promoter capable of driving expression in a plant cell acid sequence. As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant comprising a heterologous polynucleotide in its genome. Typically, the heterologous polynucleotide is stably integrated into the genome of the transgenic or transformed plant so that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant expression cassette.

It will be understood that, as used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of heterologous nucleic acid, including those originally altered in this manner. transgenes and those produced by sexual crossing or cloning from the original transgene. As used herein, the term "transgenic" does not encompass genetic ( Chromosomal or extrachromosomal) changes.

As used herein, the term "plant" includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells, and their progeny. Parts of transgenic plants fall within the scope of embodiments of the invention and include, for example, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells in plant cells. or whole plant cells in parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, etc., from which A transgenic plant or progeny thereof previously transformed with a DNA molecule of an embodiment of the present invention and thus consists at least partially of transgenic cells. The class of plants useful in the methods of the embodiments of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocots and dicots.

Although the embodiments of the present invention do not rely on a specific biological mechanism to increase the resistance of plants to plant pests, expression of the nucleotide sequences of the embodiments of the present invention in plants can lead to the production of the pesticidal proteins of the embodiments of the present invention and increased resistance of plants to plant pests. Plants of embodiments of the present invention may be used in agriculture in methods for affecting insect pests. Certain embodiments provide transformed crop plants (eg, maize plants) useful in methods for affecting insect pests (eg, Lepidopteran pests) of plants.

A "test plant or plant cell" is a plant or plant cell that has been genetically altered (e.g. transformed) for a gene of interest, or that has been inherited from a plant or cell so altered and contains the alteration . A "control", "control plant" or "control plant cell" provides a point of reference for measuring phenotypic changes in a test plant or plant cell.

A control plant or plant cell may include, for example: (a) a wild-type plant or cell, i.e., a plant or cell having the same genotype as the starting material used to make the genetic alteration that would result in a test plant or cell; (b) a plant or plant cell that has the same genotype as the starting material but has been transformed with a null construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); ( c) a plant or plant cell that is a non-transformed segregant among the progeny of the test plant or plant cell; (d) is genetically identical to the test plant or plant cell but has not been exposed to a plant or plant cell that induces expression of the gene of interest The plant or plant cell of the condition or stimulus; or (e) the test plant or plant cell itself under conditions such that the gene of interest is not expressed.

Those skilled in the art will readily recognize that advances in the field of molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, provide amino acid sequences suitable for use in the analysis of proteins of agricultural interest. An extensive collection of tools and solutions for altering or engineering both genetic sequences and corresponding genetic sequences.

Thus, the proteins of the embodiments of the present invention can be altered in various ways including amino acid substitutions, deletions, truncations and insertions. Methods of such manipulations are well known in the art. For example, amino acid sequence variants of pesticidal proteins can be prepared by introducing mutations into synthetic nucleic acids (eg, DNA molecules). Methods of mutagenesis and nucleic acid alteration are well known in the art. For example, oligonucleotide-mediated site-directed mutagenesis techniques can be used to introduce designed changes. See eg Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al., (1987) Methods in Enzymol. 154:367-382; in Molecular Biology (MacMillan Publishing Company, New York); and references cited therein.

Modifications may be made to the mutagenized nucleotide sequences of embodiments of the invention to alter about 1, 2, 3, 4, 5, 6, 8, 10, 12, or more amino acids. Alternatively, even more changes may be introduced to the native sequence such that the encoded protein may have at least about 1% or 2% or about 3%, 4%, 5%, 6% , 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22% , 23%, 24% or 25%, 30%, 35% or 40% or more of the codons are altered or otherwise modified. In the same way, the encoded protein may have at least about 1% or 2% or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, compared to the corresponding wild-type protein. %, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30 %, 35% or 40% or more additional codons. It is to be understood that the mutagenized nucleotide sequences of the embodiments of the present invention are intended to encompass biologically functionally equivalent peptides having insecticidal activity, such as improved insecticidal activity as determined by antifeedant properties against European corn borer larvae, Such sequences may arise due to codon redundancy and functional equivalence known to occur naturally within nucleic acid sequences and thus the proteins they encode.

Those skilled in the art will recognize that amino acid additions and/or substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, charge, size, and the like. Exemplary amino acid substitution sets that take into account each of the foregoing features are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine amides; and valine, leucine, and isoleucine.

Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the models described in: Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), which is incorporated herein by reference. Conservative substitutions can be made, such as exchanging one amino acid for another with similar properties.

Thus, the genes and nucleotide sequences of the embodiments of the present invention include both native sequences and mutated forms. Likewise, proteins of embodiments of the present invention encompass native proteins and modified forms (eg, truncated polypeptides) and modified (eg, mutant) forms thereof. This variant will continue to have the desired insecticidal activity. Clearly, the mutations to be made in the nucleotide sequence encoding the variant must not place the sequence out of reading frame, and generally do not create complementary regions that might produce secondary mRNA structure. See EP Patent Application Publication 75,444.

Deletions, insertions and substitutions of protein sequences covered herein are not expected to result in fundamental changes in the properties of the protein. However, when it is difficult to predict the exact effect of a substitution, deletion or insertion before it is made, those skilled in the art will know that the effect will be assessed by conventional screening assays such as insect feeding assays. See, eg, Marrone et al., (1985) J. Econ. Entomol. 78:290-293 and Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485, which are incorporated herein by reference.

Variant nucleotide sequences and proteins also encompass sequences and proteins derived from mutagenic and recombinogenic procedures such as DNA shuffling. Using this procedure, one or more different coding sequences can be manipulated to produce new pesticidal proteins with desired properties. In this manner, libraries of recombinant polynucleotides are generated from a set of related polynucleotide sequences comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this method, the full-length coding sequence of the nucleotide sequence of the embodiment of the present invention, the sequence motif encoding the domain of interest, or any fragment thereof can be combined with other known Cry Shuffles are performed between corresponding portions of the nucleotide sequence to obtain new genes encoding proteins with improved properties of interest.

Properties of interest include, but are not limited to, pesticidal activity per unit of pesticidal protein, protein stability, and toxicity to non-target species such as humans, livestock, and plants and microorganisms expressing pesticidal polypeptides of embodiments of the invention. Embodiments of the present invention are not limited to a specific shuffling strategy, as long as at least one nucleotide sequence of an embodiment of the present invention, or a portion thereof, is involved in this shuffling strategy. The shuffling may involve only the nucleotide sequences disclosed herein, or may additionally involve the shuffling of other nucleotide sequences known in the art. Strategies for DNA shuffling are known in the art. See, e.g., Stemmer (1994) Proc. (1997) J.Mol.Biol.272:336-347; Zhang et al., (1997) Proc.Natl.Acad.Sci.USA 94:4504-4509; Crameri et al., (1998) Nature 391:288-291; and US Patents 5,605,793 and 5,837,458.

The nucleotide sequences of the embodiments of the present invention may also be used to isolate corresponding sequences from other organisms, particularly other bacteria, more particularly other Bacillus strains. In this manner, methods such as PCR, hybridization, etc. can be used to identify such sequences based on their sequence homology to the sequences described herein. Embodiments of the invention encompass sequences selected on the basis of their sequence identity to the complete sequences described herein or fragments thereof. Such sequences include sequences that are orthologs of the disclosed sequences. The term "orthologs" refers to genes that are derived from a common ancestral gene and that exist in different species as a result of speciation. Genes that exist in different species are considered orthologs when their nucleotide sequences and/or the protein sequences they encode have substantial identity, as defined elsewhere herein. The function of orthologs is often highly conserved across species.

In the PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are well known in the art and are disclosed in: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, New York) , hereinafter referred to as "Sambrook". See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. , (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods utilizing paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

In hybridization techniques, all or a portion of a known nucleotide sequence is used as a probe with genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a set of clones from a selected organism The presence of other corresponding nucleotide sequences hybridizes selectively. The hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and can be labeled with a detectable group such as 32P or any other detectable label. Thus, for example, probes for hybridization can be prepared by labeling synthetic oligonucleotides based on the sequences of the embodiments of the invention. Methods for preparing probes for hybridization and constructing cDNA and genomic libraries are well known in the art and disclosed in "Sambrook".

For example, the entire sequence disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to the corresponding sequence and messenger RNA. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique to the sequences of the embodiments of the invention and are generally at least about 10 or 20 nucleotides in length. Such probes can be used to amplify the corresponding Cry sequences from selected organisms by PCR. This technique can be used to isolate additional coding sequences from a desired organism, or as a diagnostic assay to determine the presence of a coding sequence in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (plaques or colonies; see eg, Sambrook).

Hybridization of such sequences can be performed under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" means that a probe will hybridize to its target sequence to a detectably higher degree (e.g., at least 2-fold, 5-fold higher than background) than to other sequences. or 10 times) conditions. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatches in the sequences, thereby detecting lower degrees of similarity (heterologous probing). Typically, probes are less than about 1000 or 500 nucleotides in length.

Typically, stringent conditions will be wherein the salt concentration is less than about 1.5 M sodium ion, usually about 0.01 to 1.0 M sodium ion concentration (or other salt), pH 7.0 to 8.3, for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (eg, greater than 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) and at 1 to 2 times SSC (20 times SSC=3.0 MNaCl/0.3M trisodium citrate) at 50-55°C. Exemplary moderately stringent conditions include hybridization in 40-45% formamide, 1.0M NaCl, 1% SDS at 37°C and washes in 0.5x to 1x SSC at 55-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C and a final wash in 0.1 times SSC at 60-65°C for at least about 20 minutes. Optionally, the wash buffer may comprise from about 0.1% to about 1% SDS. The duration of hybridization is usually less than about 24 hours, often from about 4 to about 12 hours.

The following terms are used to describe the sequence relationship between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "Percent sequence identity" and (e) "substantially identical".

(a) As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or all of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, a "comparison window" refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window, compared to a reference sequence (excluding additions or deletions), may contain Additions or deletions (i.e. gaps) so that the two sequences are optimally aligned. Typically, the comparison window is at least 20 contiguous nucleotides in length, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art recognize that to avoid high similarity to a reference sequence due to the addition of gaps in a polynucleotide sequence, gap penalties are typically introduced and subtracted from the number of matches.

Methods for aligning sequences for comparison are well known in the art. Accordingly, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al., (1981) Adv.Appl.Math.2:482; Needleman and Wunsch The global alignment algorithm of (1970) J.Mol.Biol.48:443-453; The search-local alignment method of Pearson and Lipman, (1988) Proc.Natl.Acad.Sci.85:2444-2448; Karlin and The algorithm of Altschul, (1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Comparison of sequences to determine sequence identity can be performed using computer implementations of these mathematical algorithms. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Inc. (Mountain View, California)); GCG Wisconsin Genetics Software ALIGN program (version 2.0) and GAP, BESTFIT, BLAST in version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA) (Accelrys Inc., 9685 Scranton Road, San Diego, California, USA) , FASTA and TFASTA. Alignments using these programs can be performed using default parameters. The CLUSTAL procedure is described in detail in: Higgins et al., (1988) Gene 73:237-244 (1988); Higgins et al., (1989) CABIOS 5:151-153; Corpet et al., (1988) Nucleic Acids Res 16: 10881-90; Huang et al., (1992) CABIOS 8: 155-65; and Pearson et al., (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) (supra). The ALIGN program can use a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 when comparing amino acid sequences. The BLAST program of Altschul et al. (1990) J. Mol. Biol. 215:403 (Altschul et al., 1990, Journal of Molecular Biology, Vol. 215, p. 403) is based on Karlin and Altschul (1990) supra (Ibid.) algorithm. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleotide sequences encoding proteins of embodiments of the invention. BLAST protein searches can be performed with the BLASTN program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to proteins or polypeptides of the embodiments of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389 can be used. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform iterative searches that detect distant relationships between molecules. See Altschul et al., (1997) (Altschul et al., 1997), supra. When employing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (eg, BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). Alignment can also be performed manually by inspection.

(c) "Sequence identity" or "identity" as used herein, in the context of two nucleic acid or polypeptide sequences, refers to that aspect of the two sequences that is the same when aligned for maximum correspondence over a specified comparison window. Residues. When percent sequence identity is used with respect to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with similar chemical properties (such as charge or hydrophobicity) and therefore are not will change the functional properties of the molecule. When the sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. The difference is that sequences with such conservative substitutions are said to have "sequence similarity" or "similarity". Methods of making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than full mismatches, thereby increasing the percent sequence identity. So, for example, if identical amino acids are given 1 point and non-conservative substitutions are given 0 points, conservative substitutions are given a score between 0 and 1. Scores for conservative substitutions are calculated, for example, as implemented in the program PC/GENE (Intelligenetics, Inc., Mountain View, California, USA).

(d) As used herein, "percentage of sequence identity" means a numerical value determined by comparing two optimally aligned sequences over a comparison window, in which the multinuclear sequence is compared to a reference sequence (excluding additions or deletions). The portion of the nucleotide sequence within the comparison window may contain additions or deletions (ie, gaps) in order to optimally align the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matching positions and dividing the number of matching positions by the total number of positions in the comparison window , and then multiply the result by 100 to get the percent sequence identity.

(e)(i) The term "substantially identical" to polynucleotide sequences means that the polynucleotides contain at least 70%, when compared to a reference sequence using one of the described alignment programs using standard parameters, Sequences with 80%, 90% or 95% or greater sequence identity. Those skilled in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For these purposes, amino acid sequences being substantially identical generally means that the sequence identities are at least 60%, 70%, 80%, 90% or 95% or greater sequence identities.

Another indication that nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5°C lower than the _Tm for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1°C to about 20°C lower than the _Tm , depending on the desired degree of stringency as otherwise defined herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, for example, when one copy of a nucleic acid is produced using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) In the case of a peptide, the term "substantially identical" means that the peptide comprises a sequence which is at least 70%, 80%, 85%, 90%, 90%, 95% or greater sequence identity. Optimal alignment for these purposes can be performed using the global alignment algorithm of Needleman and Wunsch (1970) supra. An indication that two peptide sequences are substantially identical is that one peptide is immunoreactive with antibodies raised against the second peptide. Thus, for example, two peptides are substantially identical if they differ from a second peptide only by conservative substitutions. Peptides that are "substantially similar" share sequences as described above, except that residue positions that are not identical may differ by conservative amino acid changes.

The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. Furthermore, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences that can be employed in the methods of the embodiments of the invention to transform plants, including but Without limitation, those nucleotide constructs, molecules and sequences composed of deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Nucleotide constructs, nucleic acids and nucleotide sequences of embodiments of the present invention also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, and the like.

Another embodiment example relates to transformed organisms, such as organisms selected from plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes and algae. The transformed organism comprises: a DNA molecule according to an embodiment of the present invention, an expression cassette comprising said DNA molecule, or a vector comprising said expression cassette, which can be stably incorporated into the genome of the transformed organism.

The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to the sequences of the embodiments of the invention. As used herein, the term "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, if necessary to join two protein coding regions, contiguous and in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional genes may be provided on multiple DNA constructs.

This DNA construct is provided with multiple restriction sites to allow the insertion of Cry toxin sequences under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain a selectable marker gene.

The DNA construct will comprise, in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of an embodiment of the invention, a transcriptional and translational Termination zone (ie termination zone). The transcription initiation region (ie promoter) may be native, analogous, foreign or heterologous with respect to the host organism and/or with respect to the sequence of the embodiments of the invention. Additionally, the promoter may be a native sequence or alternatively a synthetic sequence. As used herein, the term "foreign" means that the promoter is not present in the native organism into which it is introduced. If a promoter is "foreign" or "heterologous" to a sequence of an embodiment of the invention, it means that the promoter is not the original or native promoter to which the sequence of an embodiment of the invention is operably linked. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. If the promoter is the original or native sequence, expression of the operably linked sequence is altered compared to wild-type expression, resulting in an altered phenotype.

The termination region may be native to the transcription initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source ( That is, foreign or heterologous to the promoter, the sequence of interest, the plant host, or any combination thereof).

Readily available termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Callis et al., (1991) Genes Dev. 5:141-149; Mogen et al., (1990) Plant Cell 2:1261-1272; Munroe et al., (1990) Gene 91:151-158; Ballas et al., (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. , (1987) Nucleic Acid Res. 15:9627-9639.

Nucleic acids can be optimized, as appropriate, for increased expression in the host organism. Thus, if the host organism is a plant, synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. For a discussion of host-preferred codon usage, see, eg, Campbell and Gowri, (1990) Plant Physiol. 92:1-11. For example, although the nucleic acid sequences of the embodiments of the invention are expressible in both monocot and dicot species, the sequences can be modified to account for the specific codon preferences and GC content preferences of either monocots or dicots. because these preferences have been shown to be different (Murray et al., (1989) Nucleic Acids Res. 17:477-498). Thus, maize preferred codons for specific amino acids can be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra. Methods for synthesizing plant-preferred genes are readily available in the art. See, eg, US Patents 5,380,831 and 5,436,391, and Murray et al., (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

Additional sequence modifications are known to enhance gene expression in cellular hosts. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of a sequence can be adjusted to an average level for a given cellular host, calculated by reference to known genes expressed in the host cell. As used herein, the term "host cell" refers to a cell that contains a vector and supports the replication and/or expression of the expression vector. The host cell may be a prokaryotic cell such as E. coli or a eukaryotic cell such as yeast, insect, amphibian or mammalian cell, or a monocotyledonous or dicotyledonous plant cell. An example of a monocot host cell is a maize host cell. When possible, sequences were modified to avoid predictable hairpin secondary mRNA structures.

The expression cassette may additionally contain a 5' leader sequence. Such leader sequences can act to enhance translation. Translation leader sequences are known in the art and include: picornavirus leaders, such as the EMCV leader (Encephalomyocarditis 5' UTR) (Elroy-Stein et al., (1989) Proc. Natl. Acad. Sci .USA 86: 6126-6130); Potato virus Y leader sequence, such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene 165 (2): 233-238); MDMV leader sequence (maize dwarf mosaic virus); human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature 353:90-94); untranslated leader from coat protein mRNA (AMV RNA 4) of alfalfa mosaic virus sequence (Jobling et al., (1987) Nature 325:622-625); Tobacco mosaic virus leader sequence (TMV) (Gallie et al. (1989), in: Molecular Biology of RNA, Cech ed. (Liss, New York), pp. pp. 237-256); and the Maize Chlorotic Mottle Virus Leader Sequence (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the multiple DNA fragments can be manipulated to provide the DNA sequence in the correct orientation and, where appropriate, in the correct reading frame. For this purpose, adapters or linkers may be used to join the DNA fragments together, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction digestion, annealing, resubstitutions (eg transitions and transversions) may be involved.

A variety of promoters are useful in the practice of embodiments of the present invention. A promoter can be chosen based on the desired outcome. The nucleic acid may be combined with a constitutive, tissue-preferred, inducible or other promoter for expression in the host organism. Constitutive promoters suitable for use in plant host cells include core promoters such as the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent 6,072,050; the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-812); Rice actin (McElroy et al., (1990) PlantCell 2:163-171); Ubiquitin (Christensen et al., (1989) Plant Mol.Biol.12:619-632, and Christensen et al., (1992) Plant Mol.Biol.18:675-689); pEMU (Last et al., (1991) Theor.Appl.Genet.81:581-588); MAS (Velten et al., (1984 ) EMBO J.3:2723-2730); ALS promoter (US Patent 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in the following US Patents: 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;

Depending on the desired outcome, it may be advantageous to express the gene from an inducible promoter. Of particular interest for modulating expression in plants of nucleotide sequences of embodiments of the invention are damage-inducible promoters. This damage-inducible promoter responds to damage caused by insect feeding and includes the potato protease inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al., (1996 ) Nature Biotechnology 14:494-498); wun1 and wun2, US Pat. 1992) Science 225:1570-1573); WIP1 (Rohmeier et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al., (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. et al., (1994) Plant J. 6(2):141-150); et al., these patents and documents are incorporated herein by reference.

Additionally, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenicity-associated proteins (PR proteins) that are induced upon pathogen infection; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitin Enzymes, etc. See, eg, Redolfi et al., (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al., (1992) Plant Cell 4: 645-656; and Van Loon, (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, which is incorporated herein by reference.

Promoters that are expressed locally at or near the site of pathogen infection are of interest. See, eg, Marineau et al., (1987) Plant Mol. Biol. 9:335-342; Matton et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al., (1986) Proc. Natl. Acad Sci. USA 83: 2427-2430; Somsisch et al., (1988) Mol. Gen. Genet. 2: 93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93: 14972-14977. See also Chen et al., (1996) Plant J. 10:955-966; Zhang et al., (1994) Proc. Natl. Acad. Sci. USA 91: 2507-2511; .3:191-201; Siebertz et al., (1989) Plant Cell 1:961-968; US Patent 5,750,386 (nematode inducible); and references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, the expression of which is induced by the pathogen Fusarium moniliforme (see, e.g., Cordero et al., (1992) Physiol. Mol. Plant Path. 41:189-200) .

Gene expression in plants is regulated by applying exogenous chemical regulators and using chemically regulated promoters. Depending on the goal, the promoter can be a chemically inducible promoter, where application of a chemical induces gene expression, or a chemically repressible promoter, where application of a chemical represses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter (which is activated by the benzenesulfonamide herbicide safener), the maize GST hydrophobic electrophilic compounds) and the tobacco PR-1a promoter (which is activated by salicylic acid). Other chemically regulated promoters of interest include steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters in Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al., (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al., (1991) Mol. Gen. Genet. 227:229-237 and US Patents 5,814,618 and 5,789,156), which are incorporated herein by reference.

Tissue-preferred promoters can be used to target enhanced pesticidal protein expression to specific plant tissues. Tissue-preferred promoters include those discussed in: Yamamoto et al., (1997) Plant J. 12(2) 255-265; Kawamata et al., (1997) Plant Cell Physiol. 38(7):792 -803; Hansen et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell et al., (1997) Transgenic Res.6(2):157-168; Rinehart et al., (1996) Plant Physiol.112(3):1331-1341; Van Camp et al., (1996) Plant Physiol.112(2):525-535; Canevascini et al., (1996) Plant Physiol.112(2):513-524; Yamamoto et al., (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al., (1993) Plant Mol Biol. 23(6) : 1129-1138; Matsuoka et al., (1993) Proc Nad. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al., (1993) Plant J. 4(3): 495-505. Such promoters can be modified, if necessary, to attenuate expression.

Leaf-preferred promoters are known in the art. See eg Yamamoto et al., (1997) Plant J.12(2):255-265; Kwon et al., (1994) Plant Physiol.105:357-67; Yamamoto et al., (1994) PlantCell Physiol.35(5 ):773-778; Gotor et al., (1993) Plant J.3:509-18; Orozco et al., (1993) Plant Mol. Biol.23(6):1129-1138; and Matsuoka et al., (1993 ) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590.

Root-preferred or root-specific promoters are known and can be selected from a number of promoters available in the literature, or isolated de novo from various compatible species. See, eg, Hire et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051- 1061 (the root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol. Biol. 14 (3): 433-443 (the mannoline synthase (MAS ) gene's root-specific promoter); and Miao et al., (1991) Plant Cell 3 (1): 11-22 (a full-length cDNA clone encoding a cytosolic glutamine synthetase (GS) in expressed in soybean roots and nodules). See also Bogusz et al., (1990) Plant Cell 2(7): 633-641, which describes the extraction from the nitrogen-fixing non-legume Ulmaceae mountain jute (Parasponia andersonii) and related non-nitrogen-fixing non-legume chicken droppings. Two root-specific promoters isolated from the hemoglobin gene of Trema tomentosa. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume plant Nicotiana tabacum and the legume plant Lotus corniculatus, and in both cases the root Specific promoter activity is maintained. Leach and Aoyagi (1991) describe the results of their analysis of the promoters of the highly expressed rolC and rolD root-inducible genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancers and tissue-preferred DNA determinants are segregated within those promoters. Teeri et al., (1989) demonstrated using a gene fusion with lacZ that the Agrobacterium T-DNA gene encoding octopine synthase is particularly active in the root apical epidermis and that the TR2' gene is root-specific in intact plants Sexual and stimulated by wounding in leaf tissue, this is a particularly desirable combination of properties for insecticidal or larvicidal genes (see EMBO J.8(2):343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar properties. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol. 29(4):759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. 25(4):681-691). See also the following US Patents: 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;

"Seed-preferred" promoters include "seed-specific" promoters (such promoters are active during seed development, such as the promoters of seed storage proteins) and "seed germination" promoters (such promoters are active during seed germination). active). See Thompson et al., (1989) BioEssays 10:108, which is incorporated herein by reference. Preferred promoters for such seeds include, but are not limited to, Cim1 (cytokinin-inducible message); cZ19B1 (maize 19 kDa zein); and milps (inositol-1-phosphate synthase) (see U.S. Patent 6,225,529, which literature is incorporated herein by reference). γ-Zein and Glob-1 are endosperm-specific promoters. For dicotyledonous plants, seed-specific promoters include, but are not limited to, the bean β-phaseolin gene promoter, rapeseed protein (napin) gene promoter, β-conglycin gene promoter, soybean lectin gene promoter , cruciferous protein gene promoter, etc. For monocots, seed-specific promoters include, but are not limited to, maize 15kDa zein, 22kDa zein, 27kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1 etc. See also WO 00/12733, which discloses seed-preferred promoters from the end1 and end2 genes; incorporated herein by reference. A promoter having "preferred" expression in a particular tissue is expressed to a greater extent in that tissue than in at least one other plant tissue. Some tissue-preferred promoters are expressed almost exclusively in specific tissues.

If low-level expression is desired, a weak promoter should be used. In general, as used herein, the term "weak promoter" refers to a promoter that drives expression of a coding sequence at low levels. By "low level expression" is meant a level of about 1/1000 transcript to about 1/100,000 transcript to about 1/500,000 transcript. Alternatively, it is recognized that the term "weak promoter" also encompasses promoters that drive expression in only a few cells and not in others, resulting in an overall low level of expression. If the promoter drives unacceptably high levels of expression, portions of the promoter sequence may be deleted or modified to reduce expression levels.

Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and US Patent 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those described in the following US Patents: 5,608,149; 5,608,144; 5,604,121; 5,569,597;

Generally, the expression cassette will contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds that Compounds are, for example, glufosinate-ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetic acid (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to: genes encoding resistance to the following antibiotics: chloramphenicol (Herrera Estrella et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al., (1983) Nature 303:209-213; and Meijer et al., (1991) Plant Mol.Biol.16:807-820); streptomycin (Jones et al., (1987) Mol.Gen .Genet.210:86-91); Spectinomycin (Bretagne-Sagnard et al., (1996) Transgenic Res.5:131-137); Bleomycin (Hille et al., (1990) Plant Mol-Biol. 7:171-176); Sulfonamide (Guerineau et al., (1990) Plant Mol. Biol. 15:127-136); Bromoxynil (Stalker et al., (1988) Science 242:419-423); Glycolic acid Phosphine (Shaw et al. (1986) Science 233:478-481; and US Patents 7,709,702 and 7,462,481); phosphinothricins (DeBlock et al. (1987) EMBO J. 6:2513-2518). See mainly: Yarranton (1992) Curr. Opin. Biotech. 3: 506-511; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318; Yao et al., (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al., (1980), In: The Operon, pp. 177-220; Hu et al., (1987) Cell 48:555-566 ; Brown et al., (1987) Cell 49:603-612; Figge et al., (1988) Cell 52:713-722; Deuschle et al., (1989) Proc.Natl.Acad.Sci.USA 86:5400-5404; Fuerst et al., (1989) Proc.Natl.Acad.Sci.USA 86:2549-2553; Deuschle et al., (1990) Science 248:480-483; Dr. Gossen (1993) University of Heidelberg, Germany Papers; Reines et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al., (1990) Mol. Cell. Biol. 10:3343-3356; USA 89:3952-3956; Baim et al., (1991) Proc.Natl.Acad.Sci.USA 88:5072-5076; Wyborski et al., (1991) Nucleic Acids Res.19: 4647-4653; Hillenand-Wissman (1989) Topics Mol.Struc.Biol.10:143-162; Degenkolb et al., (1991) Antimicrob.Agents Chemother.35:1591-1595; Kleinschnidt et al., (1988) Biochemistry27:1094 -1104; Bonin (1993) PhD dissertation, University of Heidelberg, Germany; Gossen et al., (1992) Proc.Natl.Acad.Sci.USA 89:5547-5551; Oliva et al. People, (1992) Antimicrob.AgentsChemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology ("((Handbook of Experimental Pharmacology "), Vol. 78 (Springer-Verlag, Berlin); and Gill et al. Man, (1988) Nature 334:721-724. These disclosures are incorporated herein by reference.

The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in embodiments of the present invention.

Methods of embodiments of the invention involve introducing polypeptides or polynucleotides into plants. "Introducing" is intended to mean presenting a polynucleotide or polypeptide to a plant in such a way that the polynucleotide or polypeptide sequence enters the interior of the plant cell. The methods of the embodiments of the present invention do not depend on the specific method of introducing the polynucleotide or polypeptide into the plant, as long as the polynucleotide or polypeptide can enter the interior of at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated transformation methods.

"Stable transformation" means that a nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means the introduction of a polynucleotide into a plant without it integrating into the plant's genome, or the introduction of a polypeptide into a plant.

Transformation protocols, and protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell (ie, monocot or dicot) targeted for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include: microinjection (Crossway et al., (1986) Biotechniques 4:320-334), electroporation (Riggs et al., (1986) Proc. USA 83:5602-5606), Agrobacterium-mediated transformation (US Pat. Particle acceleration (see, e.g., U.S. Patents 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, Gamborg and Phillips eds. (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). For potato transformation, see Tu et al., (1998) Plant Molecular Biology 37:829-838; and Chong et al., (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onions); Christou et al., (1988 ) Plant Physiol.87:671-674 (soybean); McCabe et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro CellDev.Biol.27P:175-182 (Soybean); Singh et al., (1998) Theor.Appl. Genet. 96:319-324 (Soybean); Datta et al., (1990) Biotechnology 8:736-740 (Rice); Klein et al., (1988) Proc.Natl.Acad.Sci.USA 85:4305-4309 (maize); Klein et al., (1988) Biotechnology 6:559-563 (maize); US Patent 5,240,855; 5,322,783 and 5,324,646; Plant Physiol. 91:440-444 (maize); Fromm et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311:763-764; U.S. Patent 5,736,369 (cereals); Bytebier et al, (1987) Proc. (Longman, New York), pp. 197-209) (Pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418, and Kaeppler et al., (1992) Theor.Appl.Genet.84 :560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda et al., (1996) Nature Biotechnology 14:745-750 (maize, by Agrobacterium tumefaciens); These documents and patents are incorporated herein by reference.

In specific embodiments, sequences of embodiments of the invention can be provided to plants using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, introducing Cry toxin proteins or variants and fragments thereof directly into plants, or introducing Cry toxin transcripts into plants. Such methods include, for example, microinjection or particle bombardment. See eg Crossway et al., (1986) Mol Gen. Genet. 202: 179-185; Nomura et al., (1986) Plant Sci. 44: 53-58; Hepler et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush et al., (1994) The Journal of Cell Science 107:775-784; all of which are incorporated herein by reference. Alternatively, Cry toxin polynucleotides can be transiently transformed into plants using techniques known in the art. Such techniques include viral vector systems, and precipitation of polynucleotides in a manner that avoids subsequent release of the DNA. Thus, transcription can occur from particle-bound DNA, but its release for integration into the genome is greatly reduced. Such methods include the use of particles coated with polyethyleneimine (PEI; Sigma #P3143).

Methods for targeted insertion of polynucleotides at specific locations in the plant genome are known in the art. In one embodiment, insertion of a polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See eg WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all of which are incorporated herein by reference. Briefly, polynucleotides of embodiments of the invention can be contained within a transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site flanked by two non-identical recombination sites corresponding to said sites of the transfer cassette. Appropriate recombinases are provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thus integrated at a specific chromosomal location in the plant genome.

Transformed cells can be grown into plants in a conventional manner. See, eg, McCormick et al., (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed line or with different lines and the resulting hybrids identified with constitutive or inducible expression of the desired phenotypic characteristic. Two or more generations can be grown to ensure stable maintenance and inheritance of expression of the desired phenotypic trait, followed by harvesting of the seeds to ensure that expression of the desired phenotypic trait has been achieved.

Nucleotide sequences of embodiments of the present invention can be provided to plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating the nucleotide construct of interest into a viral DNA or RNA molecule. It will be appreciated that recombinant proteins of embodiments of the present invention may be initially synthesized as part of a viral polyprotein, which may later be processed by in vivo or in vitro proteolysis to yield the desired pesticidal protein. It is also recognized that such viral polyproteins comprising at least a portion of the amino acid sequence of the pesticidal proteins of embodiments of the present invention may possess the desired pesticidal activity. Such viral polyproteins and their encoding nucleotide sequences are encompassed by embodiments of the present invention. Methods of providing a plant with a nucleotide construct and producing the encoded protein in the plant (involving viral DNA or RNA molecules) are known in the art. See, eg, US Patents 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; incorporated herein by reference.

Embodiments of the present invention also relate to plant propagation material of transformed plants of embodiments of the present invention, including but not limited to seeds, tubers, bulbs, bulbs, leaves, and cuttings of roots and shoots.

Embodiments of the present invention can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea) ), especially those Brassica species that can be used as a source of seed oil; alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g. pearl millet (Pennisetum glaucum), Yellow rice (Panicum miliaceum), Millet (Setaria italica), Dragongrass (Eleusine coracana)), Sunflower (Helianthus annuus), Safflower (Carthamus tinctorius), Wheat (Triticum aestivum), Soybean (Glycine max ), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachishypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee ( Coffea spp.), coconut (Cocosnucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), Guava (Psidium guajava), Mango (Mangifera indica), Olive (Olea europaea), Papaya (Carica papaya), Cashew (Anacardium occidentale), Macadamia integrifolia, Apricot (Prunus amygdalus), Sugar Beet (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetables, ornamentals and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuces (such as Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Sweet Pisum (Lathyrus) species), and members of the genus Cucumis such as cucumbers ( C. sativus), muskmelon (C. cantalupensis) and muskmelon (C. melo). Ornamental plants include rhododendrons (Rhododendron species), hydrangea (Macrophyllahydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa species), tulips (Tulipa species), narcissus (Narcissus (Narcissus species), petunia (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Conifers useful in the practice of embodiments of the present invention include, for example, pine trees such as loblolly pine (Pinus taeda), swamp pine (Pinus elliotii), yellow pine (Pinus ponderosa), black pine (Pinus contorta), and Monterey pine (Pinus radiata). ; Douglas fir (Pseudotsuga menziesii); West hemlock (Tsuga canadensis); North American spruce (Picea glauca); Sequoia sempervirens; and cedars such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis). Plants of embodiments of the present invention include crop plants including, but not limited to: corn, alfalfa, sunflower, Brassica spp., soybean, cotton, safflower, peanut, sorghum, wheat, millet, Nicotiana, Sugarcane etc.

Turf grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canadian bluegrass (Poa compressa); red fescue (Festuca rubra); Grass (Agrostis palustris); Agropyrondesertorum; Agropyron cristatum; Festuca longifolia; Kentucky bluegrass (Poa pratensis); Orchard grass (Dactylis glomerata); Perennial ryegrass ( Lolium perenne); purple fescue (Festuca rubra); red-capped grass (Agrostis alba); thick-stem bluegrass (Poa trivialis); fescue (Festuca ovina); arundinacea); Timothy grass (Phleum pratense); Downy bentgrass (Agrostis canina); Puccinellia distans; Western icegrass (Agropyron smithii); Bermuda grass (Cynodon spp.); Zoysia spp.; Paspalum notatum; Axonopusaffinis; Eremochloa ophiuroides; Pennisetum clandesinum; Horsegrass (Bouteloua gracilis); Bisongrass (Buchloedactyloids); Weepweed (Bouteloua curtipendula).

Plants of interest include cereal plants (providing the seeds of interest), oilseed plants and leguminous plants. Seeds of interest include grain seeds such as corn, wheat, barley, rice, sorghum, rye, millet, and the like. Oilseed plants include cotton, soybean, safflower, sunflower, brassica, maize, alfalfa, palm, coconut, flax, castor, olive, and the like. Legumes include beans and peas. Legumes include guar beans, locust beans, fenugreek, soybeans, green beans, cowpeas, mung beans, lima beans, broad beans, lentils, chickpeas, and more.

In certain embodiments, nucleic acid sequences of embodiments of the present invention can be stacked with any combination of polynucleotide sequences of interest to produce plants having a desired phenotype. For example, the polynucleotides of the embodiments of the present invention can be stacked with any other polynucleotides encoding polypeptides having insecticidal and/or insecticidal activity, such as other Bt poisonous proteins (described in U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109), pentins (described in US Pat. No. 5,981,722), and the like. The resulting combination may also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the embodiments of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired combinations of traits, including but not limited to traits desired for animal feed such as high oil Genes (eg US Patent 6,232,529); balanced amino acids (eg hordothionins (US Patents 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson et al., (1987) Eur.J.Biochem.165:99- 106; and WO98/20122) and homomethionine proteins (Pedersen et al., (1986) J.Biol.Chem.261:6279; Kirihara et al., (1988) Gene 71:359; and Musumura et al., ( 1989) Plant Mol. Biol. 12:123)); digestibility enhancement (such as modified storage proteins (US Patent 6,858,778); and thioredoxin (US Patent 7,009,087); the disclosures of these documents and patents are incorporated by reference way incorporated into this article.

Polynucleotides of embodiments of the invention may also be stacked with traits required for disease or insecticide resistance (e.g., fumonisin detoxification genes (US Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al., (1994) Science 266:789; Martin et al., (1993) Science 262:1432; and Mindrinos et al., (1994) Cell 78:1089); acetolactate synthase (ALS) mutants conferring herbicide resistance, such as S4 and/or Hra mutations; inhibitors of glutamine synthase such as glufosinate or basta (e.g., the bar gene); and glyphosate resistance (EPSPS gene and GAT gene, as described in US Pat. Disclosure; and traits desired for processing or process products, such as high oil (eg US Patent 6,232,529); modified oil (eg fatty acid desaturase gene (US Patent 5,952,544; WO94/11516)); modified starch (eg ADPG coke Phosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme (SDBE)); and polymers or bioplastics (eg US Patent 5,602,321; β-ketothiolase , polyhydroxybutyrate synthase and acetoacetyl-CoA reductase (Schubert bacteria, (1988) J.Bacteriol.170:5837-5847) are conducive to the expression of polyhydroxyalkanoate (PHA)), the above disclosure Incorporated herein by reference.Polynucleotides of embodiments of the invention can also be used to provide agronomic traits such as male sterility (see, for example, U.S. Patent 5.583,210), stem strength, flowering time, or such as cell cycle regulation. Or gene targeting (eg WO 99/61619; WO 00/17364; WO 99/25821) and other polynucleotide combinations for transforming technical traits, the above disclosures are incorporated herein by reference.

In some embodiments, the traits of the stack may be regulatory approved traits or events, which are well known to those skilled in the art and can be found at the Center for Environmental Risk Assessment (cera-gmc.org/?action=gm_crop_database, www prefix may be used access it) and the International Service for the Acquisition of Agri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp, which can be accessed using the www prefix).

Combinations of these stacks can be produced by any method, including but not limited to by any conventional method or Methods or genetic transformation to cross-breed plants. If the traits are stacked by genetically transforming plants, the polynucleotide sequences of interest may be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as a target to introduce additional traits by subsequent transformation. A co-transformation protocol can be used to introduce the trait simultaneously with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences are to be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or different promoters. In some instances, it may be desirable to introduce a transformation cassette that inhibits expression of the polynucleotide of interest. This can be combined with any combination of other suppression or overexpression cassettes to generate the desired combination of traits in plants. It is also recognized that site-specific recombination systems can be used to stack polynucleotide sequences at desired genomic locations. See eg WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all of which are incorporated herein by reference.

Compositions of embodiments of the present invention can be used to protect plants, seeds and plant products in a variety of ways. For example, the composition may be used in a method involving placing an effective amount of the pesticidal composition in the environment of a pest by a procedure selected from spraying, dusting, broadcasting, or seed coating.

Before plant propagation material (fruits, tubers, bulbs, bulbs, grains, seeds), but especially seeds, is usually treated with a protective coating comprising herbicides, insecticides, Fungicides, bactericides, nematocides, molluscicides or a mixture of several of these preparations, if necessary, further add carriers, surfactants or application-promoting adjuvants commonly used in the formulation field to provide Protection against damage caused by bacteria, fungi or animal pests. For the treatment of seeds, the protectant coating can be applied to the seeds by impregnating the tubers or grains with a liquid formulation, or by coating the seeds with a combined wet or dry formulation. In addition, other methods of application to plants are possible in special cases, for example treatment of shoots or fruits.

Plant seeds of embodiments of the invention comprising a nucleotide sequence encoding a pesticidal protein of an embodiment of the invention may be treated with a seed protectant coating comprising a seed treatment compound such as, for example, captan, Carboxin, thiram, methalaxyl, pirimiphos-methyl, and other compounds commonly used in seed treatments. In one embodiment, a seed protectant coating comprising a pesticidal composition of an embodiment of the present invention is used alone or in combination with one of the seed protectant coatings commonly used for seed treatment.

It is recognized that entomopathogenic organisms can be transformed with genes encoding insecticidal proteins. Such organisms include baculoviruses, fungi, protozoa, bacteria and nematodes.

The gene encoding the pesticidal protein of the embodiment of the present invention can be introduced into a microbial host through an appropriate vector, and the host can be applied to the environment or plants or animals. The term "introducing" in the context of inserting a nucleic acid into a cell means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated into the cell's genome (eg, chromosome, plasmid, plastid, or mitochondrial DNA), transformed into an autonomous replicon, or transiently expressed (eg, transfected mRNA).

Microbial hosts can be selected that are known to occupy the "vegetative map" (foliage, leaf map, rhizosphere and/or root surface) of one or more crops of interest. These microorganisms are selected to be able to successfully compete with wild-type microorganisms in a particular environment, to provide stable maintenance and expression of genes expressing pesticidal proteins, and ideally to provide improved protection of the pesticide from environmental degradation and Inactivate.

Such microorganisms include bacteria, algae and fungi. Of particular concern are microorganisms: for example bacteria such as Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter ), Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, especially yeasts such as Saccharomyces ), Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of particular concern are phytograph bacterial species such as: Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum , Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, woody stick Clavibacter xyli and Azotobacter vinelandii, and plant diagram yeast species such as: Rhodotorular rubra, R. glutinis, R. marina , R. aurantiaca, Cryptococcus albidus, C. difffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevistae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae and Aureobasidium pollulans. Of particular concern are pigmented microorganisms.

There are a variety of means by which a gene expressing a pesticidal protein can be introduced into a microbial host under conditions that permit stable maintenance and expression of the gene. For example, expression cassettes can be constructed that include the nucleotide construct of interest and transcriptional and translational regulatory signals operably linked to the nucleotide construct for expression of the nucleotide construct, and host A nucleotide sequence that is homologous to a sequence in an organism (by which integration will occur) and/or a replication system that is functional in the host (by which integration or stable maintenance will occur).

Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons, termination signals, and the like. See, eg, U.S. Patents 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook; Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Davis et al., eds., ( 1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press) and references cited therein.

If the pesticidal protein-containing cells are to be treated to prolong the activity of the pesticidal protein in the cell when the treated cells are applied to the environment of the target pest, suitable host cells may include prokaryotes or eukaryotes, usually Restricted to those cells that do not produce substances that are toxic to higher organisms such as mammals. However, organisms that produce substances that are toxic to higher organisms may be used if the toxin is unstable or if the level of administration is low enough to avoid any possibility of toxicity to the mammalian host. As hosts, of particular interest will be prokaryotes and lower eukaryotes such as fungi. Exemplary Gram-negative and Gram-positive prokaryotes include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella Salmonella and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae, such as Photobacterium, Fermentative Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillus family (Lactobacillaceae); Pseudomonadaceae, such as the genera Pseudomonas and Acetobacter; Azotobacteraceae; Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which include yeasts such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeasts , such as Rhodotorula, Aureobasidium, Sporobolomyces and the like.

Characteristics of particular concern when selecting host cells for pesticidal protein production include ease of introduction of the pesticidal protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and accessibility The presence of hereditary ability. Characteristics of particular concern for use as insecticide microcapsules include insecticide protective qualities such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness; ease of killing and immobilization without damage to the toxin; etc. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeasts such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp. (e.g. S. cerevisiae) , Sporobolomyces spp. species, foliar organisms such as Pseudomonas spp. species (such as P. aeruginosa, P. fluorescens ), Erwinia spp. and Flavobacterium spp. and other such organisms, including Bt, E. coli, Bacillus subtilis, and the like.

Genes encoding pesticidal proteins according to embodiments of the present invention can be introduced into microorganisms (epiparasites) that propagate on plants to deliver the pesticidal proteins to potential target pests. Epiparasites may, for example, be Gram-positive or Gram-negative bacteria.

Root-colonizing bacteria can be isolated from a plant of interest by, for example, methods known in the art. In particular, root-colonizing Bacillus cereus strains can be isolated from the roots of plants (see, eg, Handelsman et al. (1991) Appl. Environ. Microbiol. 56:713-718). The gene encoding the pesticidal protein of the embodiment of the present invention can be introduced into Bacillus cereus by standard methods known in the art.

The gene encoding the insecticidal protein can be introduced into, for example, Bacillus rhizogenes by means of electroporation. In particular, genes encoding pesticidal proteins can be cloned into a shuttle vector such as pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218). Shuttle vector pHT3101 containing the coding sequence for a specific insecticidal protein gene can be transformed, for example, by electroporation into Bacillus rhizogenes (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218).

Expression systems can be designed such that pesticidal proteins are secreted outside the cytoplasm of Gram-negative bacteria such as E. coli. The benefits of secreting a pesticidal protein are: (1) avoiding the potential cytotoxic effects of the expressed pesticidal protein; and (2) improving the purification efficiency of the pesticidal protein, including but not limited to increased protein recovery per volume of cell culture fluid and Purification efficiency and reduced recovery and purification time and/or cost per unit of protein.

Pesticidal proteins can be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino terminus of the pesticidal protein. Signal peptides recognized by E. coli may be present in proteins known to be secreted in E. coli, such as the OmpA protein (Ghrayeb et al. (1984) EMBO J, (European Molecular Biology Organization Journal) 3: 2437-2442). OmpA is the major protein of the outer membrane of E. coli, so its signal peptide is thought to be effective in the translocation process. In addition, the OmpA signal peptide does not require modification prior to processing, whereas other signal peptides such as lipoprotein signal peptides do (Duffaud et al. (1987) Meth. Enzymol. 153:492).

Insecticidal proteins of embodiments of the present invention can be fermented in bacterial hosts, and the resulting bacteria processed and used as microbial sprays in the same manner as Bt strains have been used as insecticidal sprays. In cases where the pesticidal protein is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the pesticidal protein into the growth medium during fermentation. The pesticidal protein remains inside the cell, and the cell is then processed to obtain the encapsulated pesticidal protein. Any suitable microorganism can be used for this purpose. Bt toxins have been expressed as encapsulating proteins using Pseudomonas and the resulting cells processed and sprayed as pesticides (Gaertner et al. (1993) In: Advanced Engineered Pesticides, Kim ed.).

Alternatively, the pesticidal protein is produced by introducing a heterologous gene into the cellular host. Expression of the heterologous gene leads directly or indirectly to the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the produced toxin in the cells when the cells are applied to the environment of the target pest. The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticidal proteins can then be formulated according to conventional techniques for application to the host environment of the target pest, such as soil, water and foliage of plants. See eg EP0192319 and references cited therein.

In embodiments of the present invention, transformed microorganisms (which include whole organisms, cells, spores, pesticidal proteins, pesticidal components, pest-affecting components, mutants, living or dead cells and cell components , including living or dead cells and mixtures of cell components, and including broken cells and cell components) or isolated pesticidal proteins are formulated with acceptable carriers such as the following pesticidal compositions: Suspensions, solutions, emulsions, dusting powders, dispersible granules or pellets, wettable powders and emulsifiable concentrates, aerosols or sprays, impregnated granules, adjuvants, spreadable pastes, colloids and also e.g. in polymers Encapsulations in substances. Such formulated compositions can be prepared by conventional means such as drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating a culture of cells comprising the polypeptide.

Compositions of the type disclosed above can be obtained by adding: surfactants, inert carriers, preservatives, humectants, feeding stimulants, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, buffers, glidants or fertilizers, micronutrient donors or other products that affect plant growth. One or more of herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, harvesting aids and fertilizers may be used Agrochemicals are combined with carriers, surfactants or adjuvants commonly used in formulations to facilitate product handling and application to specific target pests. Suitable carriers and auxiliaries can be solid or liquid and correspond to the substances customary in formulation technology, for example natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The active ingredients of the embodiments of the present invention are usually applied in the form of compositions and may be applied to the crop area, plants or seeds to be treated. For example, compositions of embodiments of the present invention may be applied to grain in preparation for storage or during storage in a grain silo or silo or the like. Compositions of embodiments of the present invention may be administered simultaneously or sequentially with other compounds. Methods of applying an active ingredient of an embodiment of the invention or an agrochemical composition of an embodiment of the invention comprising at least one pesticidal protein produced by a bacterial strain of an embodiment of the invention, including but not limited to application to foliage, coating of seeds and applied to the soil. The number of applications and the rate of application depend on the intensity of the respective pest infestation.

Suitable surfactants include, but are not limited to, anionic compounds such as, for example, carboxylates of metals; carboxylates of long chain fatty acids; N-acyl sarcosinates; mono- or diesters of phosphoric acid with fatty alcohol ethoxylates or Salts of such esters; fatty alcohol sulfates such as sodium lauryl sulfate, sodium stearyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates ; lignosulfonates; petroleum sulfonates; alkylarylsulfonates such as alkylbenzenesulfonates or lower alkylnaphthalenesulfonates, such as butylnaphthalenesulfonates; sulfonated naphthalene-formaldehyde condensation salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as amide sulfonates, for example the sulfonated condensation products of oleic acid and N-methyltaurine; or dialkylsulfonic acid Succinates such as sodium dioctyl succinate sulfonate. Nonionic agents include fatty acid esters, fatty alcohols, fatty acid amides, or condensation products of fatty alkyl or alkenyl substituted phenols with ethylene oxide, fatty acid esters of polyol ethers such as sorbitan fatty acid esters, such Condensation products of esters with ethylene oxide such as polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic diols such as 2,4,7,9-tetraethylene yl-5-decyne-4,7-diol, or ethoxylated acetylenic diol. Examples of cationic surfactants include, for example, aliphatic monoamines, diamines or polyamines such as acetates, naphthenates or oleates; or amine oxides of oxygen-containing amines such as polyoxyethylene alkylamines; amide-linked amines prepared by condensation of diamines or polyamines; or quaternary ammonium salts.

Examples of inert materials include, but are not limited to, inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or plant materials such as cork, powdered corn cobs, peanut husks, rice husks, and walnut husks.

Compositions according to embodiments of the present invention may be in a form suitable for direct application, or as a concentrate of a primary composition requiring dilution with an appropriate amount of water or other diluent prior to application. Insecticidal concentrations will vary with the nature of a particular formulation, specifically, depending on whether it is a concentrate or a direct application. The composition contains 1 to 98% solid or liquid inert carrier and 0 to 50% or 0.1 to 50% surfactant. These compositions will be applied at rates indicated for commercial products, eg, about 0.01 lbs to 5.0 lbs per acre when in dry form and about 0.01 pints to 10 pints per acre when in liquid form.

In another embodiment, compositions of embodiments of the present invention, as well as transformed microorganisms and pesticidal proteins, may be treated prior to formulation to prolong pesticidal activity when applied to the environment of target pests, so long as the pretreatment Insecticidal activity is harmless. Such treatment can be carried out by chemical and/or physical means, provided that the treatment does not deleteriously affect the properties of the composition. Examples of chemical agents include, but are not limited to, halogenating agents; aldehydes, such as formaldehyde and glutaraldehyde; anti-infective agents, such as benzalkonium chloride; alcohols, such as isopropanol and ethanol; and tissue fixatives such as Bouin's fixative and Helly fixative (see, eg, Humason (1967) Animal Tissue Techniques ("Animal Tissue Technique") (W.H. Freeman and Co.).

In other embodiments, it may be desirable to treat the Cry toxin polypeptide with a protease, such as trypsin, to activate the protein prior to applying the pesticidal protein compositions of embodiments of the present invention to the environment of the target pest. Methods for activating protoxins by serine proteases are well known in the art. See, eg, Cooksey (1968) Biochem. J. 6:445-454 and Carroll and Ellar (1989) Biochem. J. 261:99-105, the teachings of which are incorporated herein by reference. For example, a suitable activation protocol includes, but is not limited to, a polypeptide to be activated, such as a purified novel Cry polypeptide (for example, having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8) and trypsin at a ratio of 1/100 The protein/trypsin weight ratio was mixed in 20 nM NaHCO ₃ (pH 8) and the samples were digested at 36° C. for 3 hours.

Compositions (comprising transformed microorganisms and pesticidal proteins according to embodiments of the present invention) can be applied to the environment of insect pests by, for example, spraying, misting, dusting, dispersing, coating or pouring, introducing into the soil, or introducing into the soil Seed treatment or general application or loose dusting as a protective measure when pests have begun to appear or before they are introduced into irrigation water. For example, pesticidal proteins and/or transformed microorganisms of embodiments of the present invention can be mixed with grain to protect the grain during storage. It is often important to get good pest control during the early stages of plant growth, as this is when the plant is likely to suffer the most damage. Compositions of embodiments of the present invention may conveniently contain another insecticide if deemed necessary. In one embodiment, the composition is applied directly to the soil at the time of planting in the form of granules of the composition of the carrier and the Bacillus strain or the dead cells of the transformed microorganism of the embodiments of the present invention. Another embodiment is in the granular form of a composition comprising an agricultural chemical such as a herbicide, insecticide, fertilizer, an inert carrier, and dead cells of a Bacillus strain or a transformed microorganism of an embodiment of the invention.

Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of embodiments of the present invention exhibit activity against insect pests which may include economically important agricultural pests, forest pests, greenhouse pests, nursery pests, ornamental plant pests, food and fiber pests, public health and animal health pests , household and commercial facility pests, living room pests and storage pests. Insect pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Trichophaera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermatoptera, Isoptera Orders, Liquitas, Fleas, Trichoptera, etc., especially Coleoptera and Lepidoptera.

Insects of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and cotton bollworms in the family Noctuidae: Agrotis ipsilon Hufnagel (black cutworm); gray cutworm (A. orthogonia Morrison ) (western cutworm); A.segetum Denis & Schiffermüller (yellow cutworm); A.subterranea Fabricius (A.subterranea Fabricius) (ground silkworm); Alabama argillacea Hübner (Alabama argillacea Hübner) (cotton leafworm); Anticarsia gemmatalis Hübner (spiny bean caterpillar); Athetismindara Barnes and McDunnough (ground silkworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoamessoria Harris (darksided cutworm); Helicoverpa armigera Hübner (American cutworm) American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm) ); Hypena scabra Fabricius (green cloverworm); Mamestra configurata Walker (Shawl Armyworm); M. brassicae Linnaeus (cabbagemoth); Melanchra picta Harris (zebra caterpillar) ; common armyworm (Pseudaletia unipuncta Haworth) (army insect); soybean armyworm (Pseudoplusia includesensWalker) (soybean inchworm); Richia albicosta Smith (western bean armyworm); grass armyworm (Spodoptera frugiperda JE S mith) (Fall Armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (Spodoptera litura, tea worm); Trichoplusia ni Hübner (cabbage looper); from the family Pyralidae ) and borers, sheath moths, web-spinning caterpillars, pine cone moths and leaf carving insects of the family Crambidae, such as Achroia grisella Fabricius (small wax moth); navel orange moth (Amyelois transitella Walker) (naval orangeworm ); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilopartellus Swinhoe (spotted stalk borer); C. suppressalis Walker (striped stem/rice borer); C. terrenellus Pagenstecher (sugarcane two point borer); Corcyra cephalonica Stainton (ricemoth )); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (grass moth); leaf roller)); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (Melon borer); Cucumber silk borer (D.nitidalis Stoll) ( pickle worm); Diatraea grandiosella Dyar (Southwest corn borer), D. saccharalis Fabricius (sugar cane borer); South American corn seedling borer (Elasmopalpus lignosellus Zeller) (lesser cornstalk borer); Mexican rice borer (Eoreuma loftiniDyar)(Mexican rice borer); tobacco powder Ephestia elutella Hübner (tobacco (cocoa) moth); Galleria mellonella Linnaeus (greater wax moth); Hedyleptaaccepta Butler (sugarcane leaf roller); Rice leafcutter (Herpetogramma licarsisalis Walker) (meadow borer); Homoeosoma electellum Hulst (sunflower borer); Loxostegesticticalis Linnaeus (beet net borer); Cowpea pod borer (Maruca testulalis Geyer) (bean pod borer); Tea tree borer (Orthaga thyrisalis Walker) (tea tree web moth); Ostrinia nubilalis Hübner (European corn borer); Indian meal moth (Plodia interpunctella Hübner) (Indian meal moth); Scirpophaga incertulas Walker ( three-colored borer); celery net borer (Udea rubigalis Guenée) (celery leafier); and leaf rollers, aphids, seed worms, and fruit insects of the family Tortricidae, western black-headed tortrix ( Tortricidae Acleris gloverana Walsingham) (Western blackheaded budworm); A.variana Fernald (Eastern blackheaded budworm); Adoxophyesorana Fischer von R6sslerstamm (summer fruit tortrix moth); Archips) species, including A. argyrospila Walker (fruit tree leaf roller) and A. rosana Linnaeus (European leaf roller); Argyrotaenia spp .); Bonagota salubricola Meyrick (Brazilian apple leaf tortrix); Choristoneur spp. a spp.); Cochylis hospes Walsingham (striped sunflower borer); Cydialatiferreana Walsingham (filbertworm); codling moth (C. pomonella Linnaeus) (apple tumbler moth); grape fruit borer (Endopiza viteana Clemens) (Grape berry moth); Eupoecilia ambiguella Hübner (Grape moth); Grapholita molesta Busck (Pear moles); Lobesia botrana Denis & Schiffermüller (European grape moth) ; Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermüller (apple bud leafroller); and sunflower bud Moth (Suleima helianthana Riley) (sunflower bud moth).

Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (Autumn inchworm); Anarsia lineatella Zeller (Peach barley moth); Rhino front moth (Anisotasenatoria J.E.Smith) ( orange striped oakworm); tussah silkworm (Antheraea pernyi Guérin-Méneville) (tussah); silkworm (Bombyx mori Linnaeus) (silkworm); cotton miner (Bucculatrix thurberiella Busck) (cotton leaf perforator); ); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm looper); Bodhi looper (Erannis tiliaria Harris) (Basswood looper); Sugarcane bud moth (Erechthias flavistriata Walsingham) (Sugar cane bud moth ); Euproctis chrysorrhoea Linnaeus (brown-tailed moth); black moth (Harrisina americana Guérin-Méneville) (grape leaf eater); Heliothis subflexa Guenée; Hemileuca oliviae Cockrell (pasture caterpillar); American white moth (Hyphantria cunea Drury) (fall webworm); Keiferia lycopersicella Walsingham (Tomato beetle); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); Western hemlock looper (L. fiscellaria lugubrosa Hulst) (Western hemlock looper); Leucoma salicis Linnaeus (Gypsy moth); Lymantna dispar Linnaeus (Gypsy moth); Canopy caterpillar species (Malacosomaspp.); Tomato hornworm (Manduca q uinquemaculata Haworth) (Mangnatia hawkmoth, tomato hawkmoth); tobacco hawkmoth (M. Species (Orgyia spp.); Paleacrita vernata Peck (springcankerworm); Papilio cresphontes Cramer (Phryganidia californica Packard) (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycterblancardella Fabricius (spotted tenderiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus ( P. napi Linnaeus (green veined whitebutterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (Plutella xylostella Linnaeus); Boisduval & Leconte (Southern cabbage caterpillar); Sabulodes aegrotata Guenée (Omnivorous inchworm); Schizuraconcinna J.E.Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine tree liner caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta aboluta Meyrick (tomato leafminer) and apple nest moth (Yponomeuta padella Linnaeus) (ermine moth).

Of concern are the larvae and adults of the order Coleoptera, which includes weevils from the families Anthribidae, Bruchidae, and Curculionidae, including but not limited to: the boll weevil ( Anthonomus grandis Boheman) (cotton weevil); sunflower stem weevil (Cylindrocopturusadspersus LeConte) (sunflower stem weevi); cane root non-ear elephant (Diaprepes abbreviatus Linnaeus) (Diaprepes root weevi); clover leaf elephant (Hypera punctata Fabricius) (cloverleaf weevi); Lissorhoptrus oryzophilus Kuschel (rice water weevi); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevi); M.hemipterus sericeus Olivier (silky cane weevi); Sitophilus granarius Linnaeus (Grain elephant); S. oryzae Linnaeus (rice elephant); Red sunflower seed weevil (Smicronyx fulvus LeConte) (red sunflower seed weevi); Gray sunflower seed weevil (S. sordidus LeConte) (graysunflower seed weevi); Sphenophorus maidis Chittenden (corn weevil); New Guinea sugarcane weevil (Rhabdoscelus obscurus Boisduval) (New Guinea sugarcane weevi); flea beetles, melon beetles, rootworms, leaf beetles, potato beetles and leaf miners of the family Chrysomelidae, including but not Limited to: Chaetocnema ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer (corn flea beetle) Colaspis brunnea Fabricius (grape leaf beetle); Diabrotica barberi Smith&Lawrence (Northern corn rootworm); Cucumber eleven-star leaf beetle (D. undecimpuncta ta howardi Barber) (southern cornrootworm); D. virgifera virgifera LeConte (western corn rootworm); Colorado potato beetle (Leptinotarsa decemlineata Say) (Colorado potato beetle); black Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn beetle); Zygogramma exclamationis Fabricius (sunflower beetle); Coccinellidae), including but not limited to: Epilachnavarivestis Mulsant (Mexican bean ladybug); scarabs and other beetles from the family Scarabaeidae, including but not limited to: Antitrogus parvulus Britton (Childers cane grub); northern Cyclocephala borealis Arrow (northern masked chafer, grub); Southern round-headed rhinoceros beetle (C.immaculata Olivier) (southern masked chafer, grub); White-haired beetle (Dermolepida albohirtum Waterhouse) (Greyback cane beetle) ; Sugarcane rhinoceros beetle (Euetheola humilis rugiceps LeConte) (sugarcane beetle); Lepidiota frenchiBlackburn (French sugarcane scale beetle); carrot beetle (Tomarus gibbosus De Geer) (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister ( White grub); P. latifrons LeConte (June gill beetle); Popillia japonica Newman (Japanese scarab); Rhizotrogus majalis Razoumowsky (European scarab); carpet beetle from the family Dermestidae; from Nematodes of various families and genera: Knockhead family (El ateridae), Eleodes spp., Melanotus spp., including M. communis Gyllenhal (nematodes); Conoderus spp.; Conoderus spp. (Limonius spp.); Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetle from the family Scolytidae; Beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (long-horned beetle); and beetles from the family Buprestidae, including but not limited to Aphanisticus cochinchinaeseminulum Obenberger (leaf miner).

Adults and immatures of the order Diptera are of concern, including the leafminer Agromyzaparvicornis Loew (corn spot leafminer); chironomids, including but not limited to: Contariniasorghicola Coquillett (Sorghum midge ( sorghum midge)); Mayetiola destructor Say (Hessian fly); sunflower seed midge (Neolasioptera murtfeldtiana Felt) (sunflower seed midge)); wheat red midge (Sitodiplosis mosellana Géhin) mosquitoes); fruit flies (Tephritidae), Oscinella frit Linnaeus (eye flies); maggots, including but not limited to: Delia spp. species, including gray field species Delia platura Meigen (seed fly); D. coarctata Fallen (stalk fly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); American straw fly (Meromyza americana Fitch) (straw fly); house fly (Musca domestica Linnaeus) (housefly); stable fly (Stomoxys calcitrans Linnaeus) (stable fly); face fly, horn fly, blowfly, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; Gastrophilus spp.); Oestrus spp.; Hypoderma spp.; Chrysops spp.; Melophagus ovinus Linnaeus (lice and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; gnats (Prosimulium spp.); gnats (Simulium spp.); midges, sandflies, sciarids and other long-horned suborders (N ematocera).

Insects of interest include those of the order Hemiptera, such as, but not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Asterolecaniidae, Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae, Planthoppers Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae, Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

Agronomically important members from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (rice green bug); Acyrthisiphon pisum Harris (bean tube aphid); Adelgess pp. ( Aphis craccivora); Adelphocoris rapidus Say (rapid plant bug); Anasatrisus De Geer (Pumpkin bug); Aphis craccivora Koch (Black soybean aphid); A. fabae Scopoli (Black soybean aphid); Cotton aphid (A.gossypii Glover) (cotton aphid, melon aphid); corn root aphid (A.maidiradicisForbes) (corn root aphid); apple yellow aphid (A.pomi De Geer) (apple aphid); Spiraea aphid (A. spiraecola Patch) (leaf roll aphid); Indonesian round shield scale insect (Aulacaspis tegalensis Zehntner) (sugarcane scale); Aulacorthum solani Kaltenbach (eggplant aphid); Bemisia tabaciGennadius (bemisia tabaci, sweet potato whitefly); B. argentifolii Bellows & Perring (Silverleaf Whitefly); Blissus leucopterus leucopterus Say (Sorghum Rhizoma); Blostomatidaes pp. Cacopsyllapyricola Foerster (Pear yellow psyllid); Calocoris norvegicus Gmelin (potatocapsid bug); Chaetosiphon fragaefolii Cockerell (Strawberry aphid); Cimicidae spp.; Coreidae spp. ; square-winged bug (Corythuca gossypii Fabricius) (cotton bug); Cyrtopeltis modesta Distant (tomato bug); tobacco small bug (C.notatus Distant) (suckfly); foam cicada (Deois flavopicta ) (spittlebug); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Duplachionaspis divergens Green ( armored scale); rose apple aphid (Dysaphisplantaginea Paaserini) (rosy apple aphid); cotton bug (Dysdercus suturellus Herrich- ) (poison cottonworm); sugarcane mealybug (Dysmicoccus boninsis Kuwana) (gray sugarcanemealybug); broad bean microleafhopper (Empoasca fabae Harris) (potato green leafhopper); Eriosomalanigerum Hausmann (apple cotton aphid); Erythroneoura spp. (grape Leafhopper); Eumetopinaflavipes Muir (island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one- spotted stink bug); Graptostethus spp. (complex of seed bugs); and Hyalopterus pruni Geoffroy (Peach aphid); Icerya purchasesi Maskell (blowing scale insect); Labopidicola allii Knight (onion lace bug); Laodelphax striatellus Fallen (white planthopper); Leptoglossus corculus Say (pine leaf root bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug); Lipaphis erysimi Kaltenbach (radish aphid); pabulinus Linnaeus) (common green capsid); Lygus lineolaris Palisotde Beauvois (Lygus lineolaris Palisotde Beauvois); L.Hesperus Knight (L.pratensis Linnaeus) (common meadow bug); L.pratensis Linnaeus (common meadow bug); Macrosiphum euphorbiae Thomas (potato aphid); Aster leafhopper (Macrostelesquadrilineatus Forbes) (two-spotted leafhopper); Magicicada septendecim Linnaeus (periodic cicada); Mahanarva fimbriolata (Sugarcane moth cicada); Sorghum aphid (Melanaphis sacchari Zehntner) (sugarcane aphid); Mealbug (Melanaspis glomerata Green) (black scale); Metopolophium dirhodum Walker (rose grain aphid); Myzus persicae Sulzer) (green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); black-tailed leafhopper (Nephotettix cinticepsUhler) (green leafhopper); two black-tailed leafhopper (N. nigropictus ) (Black-tailed leafhopper); Nezaraviridula Linnaeus (Nezaraviridula Linnaeus) (Southern rice green bug); Nilaparvata lugens (Nysiusericae Schilling) (false chinch bug) Nysius raphanus Howard (false wheat bug); Oebalus pugnax Fabricius (rice brown bug); Oncopeltus fasciatus Dallas ( large milkweed longbug); Orthops campestris Linnaeus; Pemphigus spp. (root aphid and gall aphid); Peregrinus maidis Ashmead (corn planthopper); sugarcane flathorn planthopper (Perkinsiella saccharicida Kirkaldy) (sugarcane delphacid); Phylloxera devastatrix Pergande (Pylloxera americana); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsuslineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton Lygus); Pseudococcus spp. (other mealybug complexes); Cotton Grass Scale (Pulvinariaelongata Newstead ( Cotton Grass Scale); Pyrilla perpusilla Walker (sugarcaneleafhopper); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (San. Joseph bug); .); Rhopalosiphummaidis Fitch (corn leaf aphid); R. padi Linnaeus (grain aphid); Saccharicoccus sacchari Cockerell (pink sugarcane mealybug); ); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (wheat Sogatella furcifera Horvath (white-backed planthopper); Sogatodes oryzicola Muir (rice planthopper); Spanagonicus albofasciatus Reuter (Pythia spp.); Spotted alfalfa aphid (Therioaphis maculata Buckton) (Alfalfa spotted aphids); Tinidae spp.; Toxopteraaurantii Boyer de Fonscolombe (orange two-pronged aphid); and T. citricida Kirkaldy (brown orange aphid); Trialeurodes abutiloneus (whitefly) and T. vaporariorum Westwood (greenhouse Whitefly); Trioza diospyri Ashmead (Psyllium persimmon); and Typhlocybapomaria McAtee (White apple leafhopper).

Additionally, adults and larvae of the order Acari (acarids) are included, such as Aceria tosichella Keifer (theatrolling spider mites) Apple Panonychus ulmi Koch (apple spider mites); (brown wheat mite); Steneotarsonemus bancrofti Michael (Sugarcane fine mite); Tetranychus and red mites of the family Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane leaf mite) , O.pratensis Banks (meadow mite), O.stickneyi McGregor (sugarcane spider mite); Tetranychus urticae Koch (two-spotted spider mite); Mai spider mite (T.mcdanieli McGregor) (McDaniel mite); T.cinnabarinus Boisduval (red spider mite); T.turkestani Ugarov & Nikolski (strawberry spider mite), Grape brevipalpus mite (Tenuipalpidae), Brevipalpus lewisi McGregor (citrus spider mite); Rust mite and Bud ticks and other spider mites and mites important to human and animal health, i.e. dust mites of the family Epidermoptidae, demodex mites of the family Demodicidae, meal mites of the family Glycyphagidae , a tick of the family Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (the lone star tick); and itchy and scabies mites of the families Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of concern, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).

Additional arthropod pests include: spiders of the order Araneae such as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); and Latrodectus mactans Fabricius (black widow spider); and Scutigeromorpha Myriapods such as Scutigeracole optrata Linnaeus (snag). In addition, insect pests of the order Isoptera are of interest, including those of the termite family (termitidae), such as, but not limited to, Cylindrotermes nordenskioeldi Holmgren and Pseudacanthotermes militaris Hagen (sugarcane termites). Insects of the order Thysanoptera are also of interest, including but not limited to thrips such as Stenchaetothrips minutus van Deventer (Sugarcane Thrips).

Compositions of embodiments of the present invention may be tested for insecticidal activity against insect pests at an early developmental stage (eg, as larvae or other immature forms). Insects may be reared in complete darkness at about 20°C to about 30°C and about 30% to about 70% relative humidity. Bioassays can be performed as described in Czapla and Lang (1990) J. Econ. Entomol. 83(6):2480-2485. Methods of rearing insect larvae and conducting bioassays are well known to those of ordinary skill in the art.

There are a variety of bioassay techniques known to those skilled in the art. The general procedure involves adding the experimental compound or organism to a food source in a closed container. Pesticidal activity is then measured by, but not limited to, changes in mortality, weight loss, attraction, repulsion, and other behavioral and physical changes after feeding and exposure for an appropriate period of time. The bioassays described herein can be used for any feeding insect pest in either the larval or adult stages.

The following examples are given by way of illustration, not limitation of the invention.

experiment

Example 1: Gene Identification and Escherichia coli (E.coli) Expression

The gene (SEQ ID NO: 1 ) encoding the Cry1J-like insecticidal protein of SEQ ID NO: 2 (Cry1JDP166) is available from: Bacillus thuringiensis isolates screened from an internal DuPont proprietary collection of strains designated DP166.

Cloning the polynucleotide sequence encoding the Cry1J polypeptide of the present disclosure into the pET28a vector and transformed into E. coli BL21 cells (Invitrogen). Large scale 1.0 L cultures were grown until OD600nm~0.8, then cultures were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) 1 mM and grown at 16°C for 16 hours. Cell pellets were lysed with 50 mL of 500 mM NaCl/20 mM Tris/5 mM imidazole/pH 7.9 supplemented with 0.02% lysozyme (w/v) and 0.1% Tween-20 and 1 tablet of complete protease inhibitor (Roch). After lysis, the solution was sonicated and the lysate was centrifuged at 25,000 rpm for 30 minutes. The supernatant containing the soluble protein fraction was filtered through a 0.45u vacuum filter and then 1 ml of Talon (Clontech) slurry was added followed by incubation on a rotator at 100 rpm for 1 hour for binding. The lysate was then added to the column and bound protein was separated and washed with 20 ml of 50 mmM NaCl/20 mM Tris/5 mM imidazole/pH 7.9 followed by elution with 1.5 ml of 50 mmM NaCl/20 mM Tris/500 mM imidazole/pH 7.9. The purified protein was then dialyzed into 50 mM sodium carbonate buffer (pH 10). In the in vitro feeding assay, insecticidally active purified proteins are applied to Lepidoptera plates.

Example 2: Lepidoptera assay with partially purified protein

Bioassay screens for insecticidal activity were performed to evaluate insecticidal proteins against various Lepidoptera species: European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), black ground silkworm (Agrotis ipsilon), fall armyworm ( Spodoptera frugiperda), soybean looper (Pseudoplusia includens) and bean caterpillar (Anticarsia gemmatalis).

Lepidoptera feeding assays were performed on artificial diets containing purified protein in 96-well plate devices. Purified protein (25ul) was then added to artificial diet. Put 2 to 5 newborn larvae into each hole, and feed them arbitrarily for 5 days. Results are indicated as positive for larval responses such as stunting and/or death. The result was indicated as negative if the larvae were similar to the negative control fed a diet supplemented with the above buffer only. Each was tested on European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), black ground silkworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), soybean looper (Pseudoplusia includens) and bean caterpillar (Anticarsia gemmatalis). The purified proteins were screened in primary bioassays at a single concentration. Insect assays are scored on the following scale: 3 = 100% mortality; 2 = severe stunting; 1 = stunting; and 0 = no activity.

Results of preliminary insecticidal assays for Cry1JDP166 insecticidal polypeptides are shown in Table 1.

Table 1

FAW CEW SBL VBC initial screening + + + +

Embodiment 3: Mutation of Cry1JDP166 polypeptide

The Cry1JDP166 polypeptide (SEQ ID NO: 2) is unstable in a trypsin solution and in the midgut fluid of the target insect pest at a ratio (W/W) of 1:50. Several mutations were designed to stabilize the wild-type CrylJDP166 polypeptide. Surprisingly, when treated with trypsin and in the midgut fluid of the target insect pest, the mutant in which position 578 of SEQ ID NO: 2 was substituted by proline to valine (referred to as Cry1JP578V (SEQ ID NO : 4) all endow the recombinant Cry1JDP166 polypeptide with stability. The insecticidal activity of Cry1JP578V (SEQ ID NO: 4) is shown in Table 2.

Table 2

target insect pest ECB CEW FAW SBL VBC Cry1JP578V (SEQ ID NO: 4) + + + + +

Example 4: Cross-resistance to insecticidal polypeptides in Cry1 Ab or Cry1F resistant strains of European corn borer (ECB)

To determine whether Cry1Ab and Cry1F resistant insects are cross-resistant to Cry1J, Cry1Ab (Crespo A. et al., Pest Manag Sci65: 1071-1081 2009) or Cry1F susceptible or resistant insects were treated with Cry1JP578V (SEQ ID NO: 4). European corn borer (Ostrinia nubilalis) larvae.

Experimental Cry1F-selected strains (Cry1F-R) were derived from a combination of five field populations collected in 2007 and were selected for resistant strains using increasing amounts of freeze-dried leaf tissue of Cry1F-expressing maize.

Larval susceptibility of Bt-susceptible and resistant strains (CryAb-R and Cry1F-R) of the corn borer (Ostrinia nubilalis) (European corn borer) to insecticidal proteins was determined using a feed incorporation bioassay. Briefly, 25ul of sample concentrate was mixed with 75ul of artificial feed in individual wells of a 96-well plate format. In addition to negative controls, each bioassay included eight to ten sample concentrates, and each concentrate was assayed in three to four replicates. Prepare a protein solution by mixing the protein with an appropriate amount of buffer solution. One neonatal larva (<24 hours post hatch) will be placed in each assay well. After feeding the treated diet for 6 days at 27°C, 50% RH and a photoperiod of 16:8 hours (L:D), the mortality and growth inhibition of larvae caused by each sample (if the larvae did not develop within 6 days) Entering the second instar, it is defined as inhibition) for scoring. Based on the probabilistic meta-analysis, the concentration at which 50% mortality (LC50) or 50% of individuals were inhibited (IC50) was calculated and statistical analysis was performed by using statistical software.

The resistance ratio of Cry1JP578V (SEQ ID NO: 4) was determined to be about 1, indicating a lack of cross-resistance between Cry1JP578V (SEQ ID NO: 4) and Cry1Ab and Cry1F insecticidal polypeptides against European corn borer (Ostrinia cerevisiae) larvae.

The Cry1Fa FAW resistant population was also tested for the Cry1JP578V polypeptide (SEQ ID NO: 4) and the resistance ratio was determined to be about 1, indicating that there is no cross-resistance between Cry1Fa of FAW and Cry1JP578V (SEQ ID NO: 4) .

Example 5: Transient Expression in Leaf and Insect Bioassays

For soybean, an expression-optimized coding sequence was designed. In the Cry1JDP166 expression-optimized coding sequence (SEQ ID NO: 5), the modified nucleotide sequence was introduced to change the glutamic acid at position 115 to aspartic acid relative to SEQ ID NO: 2, and the alanine at position 594 amino acid was changed to valine, the lysine at position 724 was changed to the codon for isoleucine, and the resulting polypeptide was named Cry1JPS1 (SEQ ID NO: 6). The Cry1JP578V expression-optimized coding sequence (SEQ ID NO: 7) contained the modification encoding the above three amino substitutions plus the substitution of proline at position 578 to valine and was designated as Cry1JPS1P578V (SEQ ID NO: 8).

To confirm the activity of Cry1JPS1P578V (SEQ ID NO: 8), a transient expression system was used under the control of the AtUBQ10 promoter (Dav et al., (1999) Plant Mol. Biol. 40:771-782; Norris SR et al. (1993) Plant Mol. Biol. 21(5):895-906). The introduction of Agrobacterium cell suspensions into plant cells of intact tissues allows measurement or study of Agroinfiltration methods for superinfection and subsequent plant-derived transgene expression are well known in the art (Kapila et al., (1997) Plant Science 122: 101- 108). Briefly, detached leaf discs of soybean (Glycine max) were agroinfiltrated with normalized bacterial cell cultures of test and control strains. After 4 days, 2 newborn soybean armyworms (SBL) (Chrysodeixis includens), corn earworms (CEW) (Helicoverpa zea, VBC ) (Anticarsia gemmatalis) or fall armyworm (Spodoptera frugiperda) infested leaf disks. Control leaf disks were generated with Agrobacterium containing only the DsRed fluorescent marker (Clontech ^™ , 1290 Terra Bella Ave. Mountain View, CA 94043) expression vector. Leaf discs obtained from non-infiltrated plants were included as a second control. Three days after infestation, the consumption of primary leaf tissue was scored and a score from 0 to 9 was given. Transiently expressed Cry1JPS1P578V (SEQ ID NO : 8) Protected leaf discs from being consumed by infested insects, whereas complete green tissue depletion was observed for negative controls and untreated tissues (Table 3) by Western blotting using a protein produced on Cry1JaH (SEQ ID NO: 2). Antibodies confirm transient protein expression of Cry1JPS1P578V (SEQ ID NO:8).

table 3

value illustrate 1 Leaf disc consumption greater than 90% 2 Leaf discs consume 70-80% 3 Leaf discs consume 60-70% 4 Leaf discs consume 50-60% 5 Leaf disc consumption 40-50% 6 Leaf disk consumption is less than 30% 7 Leaf disc consumption is less than 10% 8 The leaf disc has only a few pinholes 9 Leaf discs not touched by insects

Example 6: Agrobacterium-mediated transformation of maize and regeneration of transgenic plants

For Agrobacterium-mediated transformation of maize with a polynucleotide sequence such as SEQ ID NO: 1, SEQ ID NO: 3, or a maize-optimized sequence, the method of Zhao can be used (US Patent 5,981,840 and PCT Patent Publication WO98/32326 ; the contents of said patent are incorporated herein by reference). Briefly, immature embryos were isolated from maize and a suspension of the embryos and Agrobacterium in which the bacterium was capable of transferring the toxin nucleotide sequence (SEQ ID NO: 1, SEQ ID NO: 3 or a maize-optimized sequence) to The contacting is carried out in the condition of at least one cell of at least one immature embryo (step 1: infection step). In this step, immature embryos can be dipped into the Agrobacterium suspension to prime the inoculation. The embryos are co-cultivated with Agrobacterium for a period of time (step 2: co-cultivation step). After this infection step, the immature embryos can be cultured on solid medium. After this co-cultivation period, an optional "rest" step is contemplated. During this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium, without addition of a selection agent for plant transformants (step 3: resting step). Immature embryos can be cultured on solid medium containing antibiotics but no selection agents, for the elimination of Agrobacterium and for the quiescent phase of infected cells. Next, the inoculated embryos are cultured on a medium containing a selection agent, and the grown transformed callus is recovered (step 4: selection step). Immature embryos are cultured on solid medium with a selection agent, resulting in selective growth of transformed cells. The callus is then regenerated into plants (step 5: regeneration step), and calli grown on selective media can be cultured on solid media to regenerate plants.

Embodiment 7: Transformation of soybean embryo

Soybean embryos are cultured with a plasmid containing the toxin nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 operably linked to an appropriate promoter as described below. bombardment. To induce somatic embryos, cotyledons 3-5 mm in length are dissected from surface-sterilized immature seeds of appropriate soybean cultivars and cultured on appropriate agar media at 26°C in the light or dark for six months. to ten weeks. The somatic embryos giving rise to the secondary embryos are then excised and placed in a suitable liquid medium. Following repeated selection of clusters of somatic embryos proliferating as early globular stage embryos, the suspension was maintained as described below.

Soybean embryogenic suspension cultures were maintained in 35 mL liquid medium on a rotary shaker at 150 rpm at 26° C. under fluorescent lights on a 16:8 hour day/night schedule. Cultures were subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

Soybean embryogenic suspension cultures are then transformed by the particle gun bombardment method (Klein et al., (1987) Nature (London) 327:70-73, US Patent 4,945,050). These transformations can be performed using a Du Pont Biolistic PDS1000/HE instrument (helium modification).

Selectable marker genes that can be used to facilitate soybean transformation include, but are not limited to: the 35S promoter from cauliflower mosaic virus (Odell et al., (1985) Nature 313:810-812), from plasmid pJR225 (from E. coli; Gritz et al. Human, (1983) Gene 25: 179-188) the hygromycin phosphotransferase gene and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. An expression cassette comprising the toxin nucleotide sequence (eg, SEQ ID NO: 1, SEQ ID NO: 3, or a maize optimized sequence) operably linked to a suitable promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension add (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1M), and 50 μL CaCl2 (2.5M). The particle preparation was then agitated for three minutes, centrifuged for 10 seconds in a microcentrifuge, and the supernatant removed. The DNA-coated particles were then washed once in 400 μL 70% ethanol and resuspended in 40 μL absolute ethanol. Sonicate the DNA/particle suspension three times for one second each. Five microliters of DNA-coated gold particles were then loaded onto each megacarrier disc.

Approximately 300-400 mg of a two-week-old suspension culture was placed in an empty 60 x 15 mm Petri dish and residual liquid was removed from the tissue with a pipette. Typically approximately 5-10 tissue plates are bombarded for each transformation experiment. The membrane burst pressure was set at 1100 psi and the chamber was evacuated to a vacuum of 28 inches of mercury. The tissue was placed approximately 3.5 inches from the retaining screen and bombarded three times. After bombardment, the tissue was bisected and returned to the liquid for culture as described above.

Five to seven days after bombardment, liquid medium was exchanged with fresh medium, and seven to twelve days after bombardment was exchanged with fresh medium containing 50 mg/mL hygromycin. The selective medium was changed weekly. Seven to eight weeks after bombardment, green, transformed tissue was observed growing out of untransformed, necrotic embryogenic clusters. Isolated green tissue was removed and inoculated into separate flasks to generate new, clonal, transformed embryogenic suspension cultures. Each new line was treated as an independent transformation event. These suspensions are then subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Embodiment 8: Regeneration of soybean somatic embryos into plants

In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.

embryo maturation

Embryos were cultured in SB196 under cool white fluorescent bulbs (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watts) at 26°C for 4-6 weeks with a photoperiod of 16: For 8 hours, the light intensity was 90-120 μE/m ² s. After this time, embryo clusters were removed to solid agar medium SB166 for 1-2 weeks. Embryo clusters were then subcultured to medium SB103 for 3 weeks. During this period, individual embryos were removed from embryo clusters and screened for ABA accumulation. It should be noted that any detectable phenotype resulting from the expression of the gene of interest can be screened for at this stage.

Embryo drying and germination

Individual mature embryos were placed in empty petri dishes (35 x 10 mM) and they were dried for approximately 4-7 days. The plate was sealed with fiber tape (creating a small wet chamber). Dried embryos were planted in SB71-4 medium, where they were allowed to germinate under the same culture conditions as described above. Germinated plantlets were removed from the germination medium and rinsed thoroughly with water, and then planted in Redi-Earth ^™ in 24-well pack trays, covered with a clear plastic cover. After 2 weeks, the hood was removed and the plants were allowed to go strong for another week. If the small plants appear to be strong, transplant them into 10" Redi-Earth ^TM trays with a maximum of 3 small plants per tray.

Leaf tissue analysis from transgenic soybean events

Leaf tissue was harvested at about V3/V4 and fed to the target Lepidoptera species. Individually with 2 newborn soybean armyworm (SBL) (Soybean leafworm), corn earworm (CEW) (Helicoverpa zea), bean armyworm (VBC) (Anticarsia gemmatalis) or Fall armyworm (Spodoptera frugiperda) infested leaf discs. Leaf discs obtained from wild-type plants were included as controls. Five days after infestation, consumption of primary leaf tissue was scored and a score from 0 to 9 was given .Expressed Cry1JPS1P578V (SEQ ID NO: 8) protected leaf discs from consumption by infested insects, while complete green tissue consumption was observed for the negative control (Table 4). Using the pair Cry1JaH (SEQ ID NO: 2) Antibody generated confirms protein expression of Cry1JPS1P578V (SEQ ID NO: 1).

Table 4

The above description of various exemplary embodiments of the present disclosure is not intended to be exhaustive or to limit the scope to the precise forms disclosed. While specific embodiments, as well as examples, are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, those skilled in the relevant art will recognize. The teachings provided herein may be adapted for purposes other than the above-described examples. Accordingly, many modifications and changes in light of the above teachings are intended to be within the scope of the appended claims.

These and other changes can be made in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and claims.

The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) cited in the Background, Detailed Description, and Examples is hereby incorporated by reference in its entirety.

Efforts have been made to ensure accuracy with respect to data used (eg, amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees Celsius; and pressure is at or near atmospheric.

sequence listing

<110> Pioneer Hi-Bred International

E.I. duPont de Nemours and Company

Abad, Andre

Dong, Hua

Rice, Janet

Shi, Xiaomei

<120> Insecticidal polypeptide with broad-spectrum activity and use thereof

<130> 5383-WO-PCT

<150> US 62/064270

<151> 2014-10-15

<160> 8

<170> PatentIn Version 3.5

<210> 1

<211> 3501

<212>DNA

<213> Bacillus thuringiensis

<400> 1

atggagatca acaatcagaa gcagtgcatc ccatacaact gtctgtctaa ccccgaagag 60

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ctcctccagt tcttgctcaa caacttcgtg ccaggaggtg gcttcatctc cggattggtt 180

gacaagatct ggggcgcatt gagaccctca gagtgggacc tgttcctcgc tcagatcgag 240

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gacaacgagg ctgcaaagtc tcgcgtgatc gacaggttcc gtatcctcga tgggcttatc 420

gaggccaaca tcccttcctt ccgtatcatc gagttcgagg tcccactgct gtccgtctat 480

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aggaccatga actctggaga caacctggaa tccgggaact tccgtactgc agggttctcc 1680

actcccttca gcttctccaa cgcacagtca accttcactc tgggtacaca gcctttctcc 1740

aaccaggagg tgtacatcga taggatcgag tttgtgccag ctgaggtgac cttcgaagcc 1800

gagtctgacc ttgagcgtgc tcagaaagcc gtgaacgcac tgttcacttc caccaaccag 1860

ctcggtctga agaccgatgt gactgactac cagatcgacc aggtctcaaa cctggttgag 1920

tgccttagtg acgagttctg cttggacgag aagcgtgagc tgtctgagaa ggtcaagcac 1980

gctaaacgcc tgtccgacaa gcgcaatctc ctgcaagacc ccaacttcac ctccatcaac 2040

cgtcaactcg atcgtggttg gcgtggatca actgacatca ccatccaagg tgggaacgac 2100

gtcttcaagg agaactacgt taccttgcca ggtaccttcg acgagtgcta ccctacctac 2160

ctgtaccaga tcatcgacga gtctaagctg aaggcctaca ctcgctatga actgagaggg 2220

tacatcgagg actcccagga cctcgaagtc tacctgatcc gctacaacgc caagcacgag 2280

accgtcaatg ttcctgggac aggatctctg tggcctctgt ccgttgagag tcctataggc 2340

agatgtggtg agcctaaccg atgcgttcct cacattgagt ggaaccctga cctggactgt 2400

tcatgtcgtg acggagagaa gtgtgctcac cactcacacc acttctcctt ggacattgac 2460

gtcggatgca ctgacctcaa tgaggacttg ggagtctggg tgatcttcaa gatcaagacc 2520

caggacggtc acgctaggtt gggtaacctc gaattcctgg aggagaagcc attgctcggt 2580

gaagcattgg ctcgcgtcaa acgcgctgag aagaagtggc gtgacaaacg tgagcagctc 2640

cagttcgaga ccaacatcgt ctacaaggag gctaaggagt cagtggatgc cctcttcgtg 2700

gactcccact acaatcgtct gcaagccgac accaacatca ccatgatcca cgctgcagac 2760

aaacgtgtcc acaggatcag ggaagcttac ttgccagagt tgtccgtgat accaggtgtg 2820

aatgctgaca tcttcgagga gctggaagga ctgatcttca cagccttcag tctctacgac 2880

gctcgcaaca tcatcaagaa cggcgacttc aacaacggtc tctcctgctg gaacgtcaag 2940

ggtcacgtgg acattcagca gaacgaccac agatctgttc tggtcgtgcc tgaatgggag 3000

tccgaggtct cacaggaagt gcgtgtttgc ccaggtagag ggtacattct cagggtgact 3060

gcttacaagg agggctatgg cgaaggatgc gtgaccatac acgagatcga ggacaacacc 3120

gacgagctga agttctccaa ctgcatcgag gaggaggttt accctactga cactggcaac 3180

gactacactg ctcaccaggg tactactgga tgtgcagacg cttgcaactc ccgtaacgtc 3240

ggctacgagg acggttacga gatcaacacc actgccagtg tcaactacaa gcctacctac 3300

gaggaggaga tgtacaccga tgtgaggagg gacaaccact gcgagtacga cagagggtat 3360

ggtaaccaca caccactgcc tgctgggtat gtgaccaagg agctggagta cttccctgaa 3420

accgacactg tgtggataga gatcggtgag actgaaggca ccttcatcgt ggattccgtc 3480

gagctgctcc ttatggagga g 3501

<210> 2

<211> 1167

<212> PRT

<213> Bacillus thuringiensis

<400> 2

Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile

20 25 30

Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp

50 55 60

Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu

65 70 75 80

Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile

85 90 95

Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala

100 105 110

Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg

115 120 125

Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Ile

130 135 140

Pro Ser Phe Arg Ile Ile Glu Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Tyr

180 185 190

Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp

195 200 205

Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln

210 215 220

Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile

245 250 255

Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser

260 265 270

Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Ile Gly Thr Leu

275 280 285

Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser

290 295 300

Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly

305 310 315 320

Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser

325 330 335

Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val

340 345 350

Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr

355 360 365

Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys

370 375 380

Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr

385 390 395 400

Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp

405 410 415

Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val

420 425 430

Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala

435 440 445

Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu

450 455 460

Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe

485 490 495

Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg

500 505 510

Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg

515 520 525

Ile Asn Gly Thr Thr Val Asn Ile Gly Asn Phe Thr Arg Thr Met Asn

530 535 540

Ser Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser

545 550 555 560

Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr

565 570 575

Gln Pro Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val

580 585 590

Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln

595 600 605

Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys

610 615 620

Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu

625 630 635 640

Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu

645 650 655

Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln

660 665 670

Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg

675 680 685

Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu

690 695 700

Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr

705 710 715 720

Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr

725 730 735

Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu

740 745 750

Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly

755 760 765

Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu

770 775 780

Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys

785 790 795 800

Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His His Phe Ser

805 810 815

Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val

820 825 830

Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly

835 840 845

Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala

850 855 860

Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu

865 870 875 880

Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp

885 890 895

Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn

900 905 910

Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu

915 920 925

Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile

930 935 940

Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp

945 950 955 960

Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Asn Gly Leu Ser Cys

965 970 975

Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser

980 985 990

Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg

995 1000 1005

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1010 1015 1020

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp

1025 1030 1035

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val

1040 1045 1050

Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr

1055 1060 1065

Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu

1070 1075 1080

Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro

1085 1090 1095

Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His

1100 1105 1110

Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala

1115 1120 1125

Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr

1130 1135 1140

Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp

1145 1150 1155

Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 3

<211> 3501

<212>DNA

<213> Artificial sequence

<220>

<223> Cry1J variant coding sequence

<400> 3

atggagatca acaatcagaa gcagtgcatc ccatacaact gtctgtctaa ccccgaagag 60

gtcctccttg atggtgagcg tatccttccc gacattgatc cactggaggt tagtctctca 120

ctcctccagt tcttgctcaa caacttcgtg ccaggaggtg gcttcatctc cggattggtt 180

gacaagatct ggggcgcatt gagaccctca gagtgggacc tgttcctcgc tcagatcgag 240

cgcttgatcg accagaggat tgaggctacc gtcagagcca aggctatcac tgagctggag 300

ggactgggta ggaactacca gatctatgcc gaggctttca aggagtggga ctctgaccct 360

gacaacgagg ctgcaaagtc tcgcgtgatc gacaggttcc gtatcctcga tgggcttatc 420

gaggccaaca tcccttcctt ccgtatcatc gagttcgagg tcccactgct gtccgtctat 480

gtccaagcag ccaacttgca cctcgcattg ctcagggact ctgtgatctt tggcgaaagg 540

tggggattga ctaccaagaa cgtgaacgac atctacaacc gccagatccg cgagatccac 600

gagtactcca accactgcgt cgacacctac aacaccgagc tggagagact cggctttcgc 660

tcaatcgctc agtggcgcat ctacaaccag ttccgtaggg agctgactct caccgtcttg 720

gacatcgtcg cactgtttcc caactacgac tctaggctgt accctatcca gaccttctcc 780

cagcttacta gggagatcgt gaccagtcca gtctcagagt tctactacgg tgtcatcaac 840

tccgggaaca tcatcggtac cctcactgag cagcagatcc gcagacctca cttgatggac 900

ttcttcaact ccatgatcat gtacacctcc gacaaccgta aggagcacta ctggagtgga 960

ctggagatga ctgcctactt taccggattc gcaggtgcac aagtgtcctt cccactggtc 1020

ggtacacgtg gagaatccgc acctccactt actgtcaggt ctgtcaacga cggcatctac 1080

cgtatcctct cagctccctt ctactcagca cccttccttg gaaccatcgt ccttggatca 1140

cgcggtgaga agttcgactt cgcactgaac aacatctccc ctccaccttc caccatctac 1200

agacaccctg gaactgtgga ctcacttgtc tctatcccac ctcaggacaa ctccgttcca 1260

cctcacagag gatcttccca ccgtctgagt cacgtgacta tgagagcctc cagtcccatc 1320

tttcactgga cccacagatc tgctaccacc accaaccacca tcaaccccaa cgccatcatc 1380

cagataccct tggtcaaggc cttcaacctg cactctggag ctacagttgt tcgcggacca 1440

ggcttcactg gtggagacat cttgaggcgt accaacaccg ggacattcgc agacatgcgt 1500

gtgaacatca ccggaccact gtctcagagg tatagggtga ggattcgcta tgccagtacc 1560

actgacctcc agttcttcac caggatcaac ggtaccaccg tcaacatcgg caacttcacc 1620

aggaccatga actctggaga caacctggaa tccgggaact tccgtactgc agggttctcc 1680

actcccttca gcttctccaa cgcacagtca accttcactc tgggtacaca ggctttctcc 1740

aaccaggagg tgtacatcga taggatcgag tttgtgccag ctgaggtgac cttcgaagcc 1800

gagtctgacc ttgagcgtgc tcagaaagcc gtgaacgcac tgttcacttc caccaaccag 1860

ctcggtctga agaccgatgt gactgactac cagatcgacc aggtctcaaa cctggttgag 1920

tgccttagtg acgagttctg cttggacgag aagcgtgagc tgtctgagaa ggtcaagcac 1980

gctaaacgcc tgtccgacaa gcgcaatctc ctgcaagacc ccaacttcac ctccatcaac 2040

cgtcaactcg atcgtggttg gcgtggatca actgacatca ccatccaagg tgggaacgac 2100

gtcttcaagg agaactacgt taccttgcca ggtaccttcg acgagtgcta ccctacctac 2160

ctgtaccaga tcatcgacga gtctaagctg aaggcctaca ctcgctatga actgagaggg 2220

tacatcgagg actcccagga cctcgaagtc tacctgatcc gctacaacgc caagcacgag 2280

accgtcaatg ttcctgggac aggatctctg tggcctctgt ccgttgagag tcctataggc 2340

agatgtggtg agcctaaccg atgcgttcct cacattgagt ggaaccctga cctggactgt 2400

tcatgtcgtg acggagagaa gtgtgctcac cactcacacc acttctcctt ggacattgac 2460

gtcggatgca ctgacctcaa tgaggacttg ggagtctggg tgatcttcaa gatcaagacc 2520

caggacggtc acgctaggtt gggtaacctc gaattcctgg aggagaagcc attgctcggt 2580

gaagcattgg ctcgcgtcaa acgcgctgag aagaagtggc gtgacaaacg tgagcagctc 2640

cagttcgaga ccaacatcgt ctacaaggag gctaaggagt cagtggatgc cctcttcgtg 2700

gactcccact acaatcgtct gcaagccgac accaacatca ccatgatcca cgctgcagac 2760

aaacgtgtcc acaggatcag ggaagcttac ttgccagagt tgtccgtgat accaggtgtg 2820

aatgctgaca tcttcgagga gctggaagga ctgatcttca cagccttcag tctctacgac 2880

gctcgcaaca tcatcaagaa cggcgacttc aacaacggtc tctcctgctg gaacgtcaag 2940

ggtcacgtgg acattcagca gaacgaccac agatctgttc tggtcgtgcc tgaatgggag 3000

tccgaggtct cacaggaagt gcgtgtttgc ccaggtagag ggtacattct cagggtgact 3060

gcttacaagg agggctatgg cgaaggatgc gtgaccatac acgagatcga ggacaacacc 3120

gacgagctga agttctccaa ctgcatcgag gaggaggttt accctactga cactggcaac 3180

gactacactg ctcaccaggg tactactgga tgtgcagacg cttgcaactc ccgtaacgtc 3240

ggctacgagg acggttacga gatcaacacc actgccagtg tcaactacaa gcctacctac 3300

gaggaggaga tgtacaccga tgtgaggagg gacaaccact gcgagtacga cagagggtat 3360

ggtaaccaca caccactgcc tgctgggtat gtgaccaagg agctggagta cttccctgaa 3420

accgacactg tgtggataga gatcggtgag actgaaggca ccttcatcgt ggattccgtc 3480

gagctgctcc ttatggagga g 3501

<210> 4

<211> 1167

<212> PRT

<213> Artificial sequence

<220>

<223> Cry1J variant

<400> 4

Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile

20 25 30

Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp

50 55 60

Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu

65 70 75 80

Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile

85 90 95

Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala

100 105 110

Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg

115 120 125

Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Ile

130 135 140

Pro Ser Phe Arg Ile Ile Glu Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Tyr

180 185 190

Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp

195 200 205

Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln

210 215 220

Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile

245 250 255

Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser

260 265 270

Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Ile Gly Thr Leu

275 280 285

Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser

290 295 300

Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly

305 310 315 320

Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser

325 330 335

Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val

340 345 350

Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr

355 360 365

Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys

370 375 380

Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr

385 390 395 400

Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp

405 410 415

Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val

420 425 430

Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala

435 440 445

Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu

450 455 460

Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe

485 490 495

Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg

500 505 510

Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg

515 520 525

Ile Asn Gly Thr Thr Val Asn Ile Gly Asn Phe Thr Arg Thr Met Asn

530 535 540

Ser Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser

545 550 555 560

Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr

565 570 575

Gln Val Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val

580 585 590

Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln

595 600 605

Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys

610 615 620

Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu

625 630 635 640

Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu

645 650 655

Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln

660 665 670

Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg

675 680 685

Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu

690 695 700

Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr

705 710 715 720

Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr

725 730 735

Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu

740 745 750

Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly

755 760 765

Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu

770 775 780

Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys

785 790 795 800

Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His His Phe Ser

805 810 815

Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val

820 825 830

Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly

835 840 845

Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala

850 855 860

Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu

865 870 875 880

Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp

885 890 895

Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn

900 905 910

Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu

915 920 925

Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile

930 935 940

Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp

945 950 955 960

Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Asn Gly Leu Ser Cys

965 970 975

Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser

980 985 990

Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg

995 1000 1005

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1010 1015 1020

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp

1025 1030 1035

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val

1040 1045 1050

Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr

1055 1060 1065

Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu

1070 1075 1080

Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro

1085 1090 1095

Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His

1100 1105 1110

Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala

1115 1120 1125

Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr

1130 1135 1140

Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp

1145 1150 1155

Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 5

<211> 3501

<212>DNA

<213> Artificial sequence

<220>

<223> soybean optimized coding sequence

<400> 5

atggagatca acaatcagaa gcaatgcatc ccatacaact gcctgtctaa cccagaggag 60

gtccttctcg acggagaacg tatactccca gacattgacc cactggaagt ctccttgtca 120

ctgctccagt tcctgctcaa caacttcgtg ccaggtggag gtttcatctc cggactcgtt 180

gacaagatct ggggagctct gagaccccagt gagtgggacc tctttcttgc tcagatcgag 240

aggctcatcg atcagaggat tgaggctaca gtcagagcca aggccatcac agagttggaa 300

ggccttggca ggaactacca gatctatgca gaagccttca aggactggga atctgatcct 360

gacaacgagg ctgcaaagtc tcgtgtcatc gacaggttta ggatactgga tggcttgatc 420

gaggctaaca tcccatcctt ccgtatcatt gagttcgagg tgcccttgct ctcagtctat 480

gtgcaagctg ccaaccttca cttggcattg ctcagagact ccgtgatctt tggcgaacgt 540

tggggcctta ccaccaagaa cgtcaacgac atctacaacc gtcagattcg cgagattcac 600

gagtattcca accactgcgt ggacacctac aacaccgagt tggaacgcct gggattcagg 660

agtatcgcac agtgggaggat ctacaaccag ttccgtcgcg aacttactct cactgtcctc 720

gatatcgtcg cactctttcc caactacgac tcacgtctgt accctatcca gaccttctcc 780

caactgactc gagagatcgt cacttcacca gtctccgagt tctactatgg agtgatcaac 840

tcaggcaaca tcatcggcac tctcacagag cagcagatcc gtagacctca cctgatggac 900

ttcttcaact ccatgatcat gtacacctct gacaaccgtc gtgagcacta ctggagtgga 960

ctggagatga cagcctactt cactggattc gcaggtgcac aggtcagttt cccactggtc 1020

ggaaccagag gtgaatctgc accacctctg actgtcaggt cagtcaacga cggtatctat 1080

cgcattctca gtgctccctt ctactcagca ccattcctcg gtaccatagt cctgggtagt 1140

agaggtgaga agttcgactt cgccctcaac aacatctcac ctcctccctc aaccatctac 1200

cgacaccctg gaactgttga ttccttggtc agtatcccac ctcaggaca ttccgttcca 1260

ccacatcgtg gctcatcaca caggctctct cacgttacca tgagggcatc tagtccaatc 1320

ttccactgga ctcacaggtc agctactacc accaaccacca tcaaccctaa cgctatcatt 1380

cagatccctt tggtgaaagc cttcaatctg cactctggtg ccacagttgt tagaggacct 1440

ggcttcacag gtggagacat actccgtagg accaacactg gaaccttcgc tgacatgcgt 1500

gtcaacatca ctggtccctt gagtcagagg tacagagtcc gcatacgtta cgcctctacc 1560

actgatctgc agttcttcac acgcatcaac ggtaccaccg tgaacattgg gaactttacc 1620

cgtaccatga actcaggcga caaccttgag tctggtaact ttaggaccgc aggtttcagt 1680

accaccttct ccttctccaa tgcccagtca aattcacct tgggtacaca acccttctcc 1740

aaccaggaag tctacattga ccgtatcgag tttgtgccag tcgaggtcac attcgaggct 1800

gaaagtgacc ttgagagagc tcagaaggca gtcaacgctc tgttcactag taccaaccag 1860

cttggactga agactgatgt gacagactat cagatcgacc aagtgtccaa cctcgttgag 1920

tgcttgtcag acgagttctg cctggacgag aaacgcgagt tgtccgagaa ggtcaagcac 1980

gctaaacgcc tctctgacaa gcgcaatctg ctccaagacc ctaacttcac atccatcaac 2040

cgtcaactcg atcgtggatg gagaggttcc accgacatca ccattcaagg aggcaacgac 2100

gtcttcaagg agaactacgt cacacttccc gggactttcg acgagtgcta tccaacctac 2160

ctctaccaga tcatcgacga gtccaagctc aaagcctaca ctcgctacga actccgtggg 2220

tacatcgagg atagtcagga cctggaggtg tatctgatcc gttacaacgc taagcacgag 2280

actgtcaatg tcccaggaac tggtagtctc tggcccttgt ccgttgagtc tcctattgga 2340

cgttgtggag aacccaatcg ctgtgtccct cacatcgagt ggaacccaga cttggattgc 2400

tcatgccgtg atggagagaa gtgcgcacac cattcacacc acttctcact ggacattgat 2460

gtcgggtgca ctgatctcaa cgaggatctt ggagtctggg tgatcttcaa gatcaagacc 2520

caggatggac acgcaaggtt gggtaacctt gagttcctgg aggagaagcc tttgctggga 2580

gaagctttgg caagggtcaa gcgtgccgag aagaagtggc gtgacaagcg tgaacagctc 2640

cagttcgaga ccaacatcgt ctacaaggag gctaaggagt cagtggacgc actgttcgtg 2700

gactctcact acaacaggtt gcaagccgac accaacatca ccatgattca cgcagctgac 2760

aagagggttc accgcattag agaggcatac ttgccagagc tctcagtcat ccctggtgtc 2820

aacgctgaca tcttcgaaga gctggaggga ctgatcttta cagccttctc cctgtacgat 2880

gctcgcaaca tcatcaagaa cggcgacttc aacaacggct tgtcctgctg gaacgtgaaa 2940

ggccacgttg atatccagca gaacgaccac agatccgtcc tggttgtccc tgaatgggaa 3000

tccgaggtta gtcaggaagt tcgcgtgtgt cctggacgtg gatacattct cagagtgacc 3060

gcttacaagg agggatacgg tgaaggatgc gtgactatcc acgagatcga ggacaacacc 3120

gacgagctca agttctccaa ctgcattgag gaggaagtgt accctactga cactgggaac 3180

gactacactg ctcaccaagg cactactgga tgtgctgatg catgcaactc tcgcaacgtt 3240

gggtacgagg atggatacga gatcaacacc acagcctccg tcaactacaa gcctacctac 3300

gaagaggaga tgtacaccga cgttaggcgt gacaaccact gtgagtacga tcgtggatac 3360

ggcaaccaca ctccattgcc agctggttat gtcaccaagg agctcgagta cttcccagag 3420

accgatactg tctggatcga gataggagag actgagggta ccttcatcgt tgacagtgtc 3480

gaactcttgc tgatggagga g 3501

<210> 6

<211> 1167

<212> PRT

<213> Artificial sequence

<220>

<223> Cry1J variant

<400> 6

Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile

20 25 30

Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp

50 55 60

Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu

65 70 75 80

Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile

85 90 95

Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala

100 105 110

Phe Lys Asp Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg

115 120 125

Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Ile

130 135 140

Pro Ser Phe Arg Ile Ile Glu Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Tyr

180 185 190

Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp

195 200 205

Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln

210 215 220

Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile

245 250 255

Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser

260 265 270

Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Ile Gly Thr Leu

275 280 285

Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser

290 295 300

Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly

305 310 315 320

Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser

325 330 335

Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val

340 345 350

Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr

355 360 365

Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys

370 375 380

Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr

385 390 395 400

Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp

405 410 415

Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val

420 425 430

Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala

435 440 445

Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu

450 455 460

Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe

485 490 495

Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg

500 505 510

Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg

515 520 525

Ile Asn Gly Thr Thr Val Asn Ile Gly Asn Phe Thr Arg Thr Met Asn

530 535 540

Ser Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser

545 550 555 560

Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr

565 570 575

Gln Pro Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val

580 585 590

Pro Val Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln

595 600 605

Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys

610 615 620

Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu

625 630 635 640

Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu

645 650 655

Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln

660 665 670

Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg

675 680 685

Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu

690 695 700

Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr

705 710 715 720

Leu Tyr Gln Ile Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr

725 730 735

Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu

740 745 750

Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly

755 760 765

Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu

770 775 780

Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys

785 790 795 800

Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His His Phe Ser

805 810 815

Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val

820 825 830

Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly

835 840 845

Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala

850 855 860

Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu

865 870 875 880

Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp

885 890 895

Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn

900 905 910

Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu

915 920 925

Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile

930 935 940

Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp

945 950 955 960

Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Asn Gly Leu Ser Cys

965 970 975

Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser

980 985 990

Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg

995 1000 1005

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1010 1015 1020

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp

1025 1030 1035

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val

1040 1045 1050

Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr

1055 1060 1065

Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu

1070 1075 1080

Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro

1085 1090 1095

Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His

1100 1105 1110

Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala

1115 1120 1125

Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr

1130 1135 1140

Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp

1145 1150 1155

Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 7

<211> 3501

<212>DNA

<213> Artificial sequence

<220>

<223> soybean optimized coding sequence

<400> 7

atggagatca acaatcagaa gcaatgcatc ccatacaact gcctgtctaa cccagaggag 60

gtccttctcg acggagaacg tatactccca gacattgacc cactggaagt ctccttgtca 120

ctgctccagt tcctgctcaa caacttcgtg ccaggtggag gtttcatctc cggactcgtt 180

gacaagatct ggggagctct gagaccccagt gagtgggacc tctttcttgc tcagatcgag 240

aggctcatcg atcagaggat tgaggctaca gtcagagcca aggccatcac agagttggaa 300

ggccttggca ggaactacca gatctatgca gaagccttca aggactggga atctgatcct 360

gacaacgagg ctgcaaagtc tcgtgtcatc gacaggttta ggatactgga tggcttgatc 420

gaggctaaca tcccatcctt ccgtatcatt gagttcgagg tgcccttgct ctcagtctat 480

gtgcaagctg ccaaccttca cttggcattg ctcagagact ccgtgatctt tggcgaacgt 540

tggggcctta ccaccaagaa cgtcaacgac atctacaacc gtcagattcg cgagattcac 600

gagtattcca accactgcgt ggacacctac aacaccgagt tggaacgcct gggattcagg 660

agtatcgcac agtgggaggat ctacaaccag ttccgtcgcg aacttactct cactgtcctc 720

gatatcgtcg cactctttcc caactacgac tcacgtctgt accctatcca gaccttctcc 780

caactgactc gagagatcgt cacttcacca gtctccgagt tctactatgg agtgatcaac 840

tcaggcaaca tcatcggcac tctcacagag cagcagatcc gtagacctca cctgatggac 900

ttcttcaact ccatgatcat gtacacctct gacaaccgtc gtgagcacta ctggagtgga 960

ctggagatga cagcctactt cactggattc gcaggtgcac aggtcagttt cccactggtc 1020

ggaaccagag gtgaatctgc accacctctg actgtcaggt cagtcaacga cggtatctat 1080

cgcattctca gtgctccctt ctactcagca ccattcctcg gtaccatagt cctgggtagt 1140

agaggtgaga agttcgactt cgccctcaac aacatctcac ctcctccctc aaccatctac 1200

cgacaccctg gaactgttga ttccttggtc agtatcccac ctcaggaca ttccgttcca 1260

ccacatcgtg gctcatcaca caggctctct cacgttacca tgagggcatc tagtccaatc 1320

ttccactgga ctcacaggtc agctactacc accaaccacca tcaaccctaa cgctatcatt 1380

cagatccctt tggtgaaagc cttcaatctg cactctggtg ccacagttgt tagaggacct 1440

ggcttcacag gtggagacat actccgtagg accaacactg gaaccttcgc tgacatgcgt 1500

gtcaacatca ctggtccctt gagtcagagg tacagagtcc gcatacgtta cgcctctacc 1560

actgatctgc agttcttcac acgcatcaac ggtaccaccg tgaacattgg gaactttacc 1620

cgtaccatga actcaggcga caaccttgag tctggtaact ttaggaccgc aggtttcagt 1680

acacccttct ccttctccaa tgcccagtca aattcacct tgggtacaca agtcttctcc 1740

aaccaggaag tctacattga ccgtatcgag tttgtgccag tcgaggtcac attcgaggct 1800

gaaagtgacc ttgagagagc tcagaaggca gtcaacgctc tgttcactag taccaaccag 1860

cttggactga agactgatgt gacagactat cagatcgacc aagtgtccaa cctcgttgag 1920

tgcttgtcag acgagttctg cctggacgag aaacgcgagt tgtccgagaa ggtcaagcac 1980

gctaaacgcc tctctgacaa gcgcaatctg ctccaagacc ctaacttcac atccatcaac 2040

cgtcaactcg atcgtggatg gagaggttcc accgacatca ccattcaagg aggcaacgac 2100

gtcttcaagg agaactacgt cacacttccc gggactttcg acgagtgcta tccaacctac 2160

ctctaccaga tcatcgacga gtccaagctc aaagcctaca ctcgctacga actccgtggg 2220

tacatcgagg atagtcagga cctggaggtg tatctgatcc gttacaacgc taagcacgag 2280

actgtcaatg tcccaggaac tggtagtctc tggcccttgt ccgttgagtc tcctattgga 2340

cgttgtggag aacccaatcg ctgtgtccct cacatcgagt ggaacccaga cttggattgc 2400

tcatgccgtg atggagagaa gtgcgcacac cattcacacc acttctcact ggacattgat 2460

gtcgggtgca ctgatctcaa cgaggatctt ggagtctggg tgatcttcaa gatcaagacc 2520

caggatggac acgcaaggtt gggtaacctt gagttcctgg aggagaagcc tttgctggga 2580

gaagctttgg caagggtcaa gcgtgccgag aagaagtggc gtgacaagcg tgaacagctc 2640

cagttcgaga ccaacatcgt ctacaaggag gctaaggagt cagtggacgc actgttcgtg 2700

gactctcact acaacaggtt gcaagccgac accaacatca ccatgattca cgcagctgac 2760

aagagggttc accgcattag agaggcatac ttgccagagc tctcagtcat ccctggtgtc 2820

aacgctgaca tcttcgaaga gctggaggga ctgatcttta cagccttctc cctgtacgat 2880

gctcgcaaca tcatcaagaa cggcgacttc aacaacggct tgtcctgctg gaacgtgaaa 2940

ggccacgttg atatccagca gaacgaccac agatccgtcc tggttgtccc tgaatgggaa 3000

tccgaggtta gtcaggaagt tcgcgtgtgt cctggacgtg gatacattct cagagtgacc 3060

gcttacaagg agggatacgg tgaaggatgc gtgactatcc acgagatcga ggacaacacc 3120

gacgagctca agttctccaa ctgcattgag gaggaagtgt accctactga cactgggaac 3180

gactacactg ctcaccaagg cactactgga tgtgctgatg catgcaactc tcgcaacgtt 3240

gggtacgagg atggatacga gatcaacacc acagcctccg tcaactacaa gcctacctac 3300

gaagaggaga tgtacaccga cgttaggcgt gacaaccact gtgagtacga tcgtggatac 3360

ggcaaccaca ctccattgcc agctggttat gtcaccaagg agctcgagta cttcccagag 3420

accgatactg tctggatcga gataggagag actgagggta ccttcatcgt tgacagtgtc 3480

gaactcttgc tgatggagga g 3501

<210> 8

<211> 1167

<212> PRT

<213> Artificial sequence

<220>

<223> Cry1J variant

<400> 8

Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile

20 25 30

Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp

50 55 60

Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu

65 70 75 80

Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile

85 90 95

Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala

100 105 110

Phe Lys Asp Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg

115 120 125

Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Ile

130 135 140

Pro Ser Phe Arg Ile Ile Glu Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Tyr

180 185 190

Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp

195 200 205

Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln

210 215 220

Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile

245 250 255

Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser

260 265 270

Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Ile Gly Thr Leu

275 280 285

Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser

290 295 300

Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly

305 310 315 320

Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser

325 330 335

Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val

340 345 350

Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr

355 360 365

Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys

370 375 380

Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr

385 390 395 400

Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp

405 410 415

Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val

420 425 430

Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala

435 440 445

Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu

450 455 460

Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe

485 490 495

Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg

500 505 510

Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg

515 520 525

Ile Asn Gly Thr Thr Val Asn Ile Gly Asn Phe Thr Arg Thr Met Asn

530 535 540

Ser Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser

545 550 555 560

Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr

565 570 575

Gln Val Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val

580 585 590

Pro Val Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln

595 600 605

Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys

610 615 620

Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu

625 630 635 640

Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu

645 650 655

Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln

660 665 670

Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg

675 680 685

Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu

690 695 700

Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr

705 710 715 720

Leu Tyr Gln Ile Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr

725 730 735

Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu

740 745 750

Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly

755 760 765

Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu

770 775 780

Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys

785 790 795 800

Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His His Phe Ser

805 810 815

Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val

820 825 830

Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly

835 840 845

Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala

850 855 860

Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu

865 870 875 880

Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp

885 890 895

Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn

900 905 910

Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu

915 920 925

Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile

930 935 940

Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp

945 950 955 960

Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Asn Gly Leu Ser Cys

965 970 975

Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser

980 985 990

Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg

995 1000 1005

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1010 1015 1020

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp

1025 1030 1035

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val

1040 1045 1050

Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr

1055 1060 1065

Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu

1070 1075 1080

Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro

1085 1090 1095

Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His

1100 1105 1110

Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala

1115 1120 1125

Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr

1130 1135 1140

Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp

1145 1150 1155

Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

Claims (26)

1. A variant Cry1J polypeptide comprising an amino acid sequence corresponding to the 578th residue of SEQ ID NO: 2 with a valine substitution, wherein the variant Cry1J polypeptide is compared to SEQ ID NO: 2 Cry1J polypeptides have enhanced stability against proteolytic degradation. 2. The variant CrylJ polypeptide of claim 1, wherein said enhanced stability is measured in a simulated gastric fluid (SGF) assay. 3. The variant CrylJ polypeptide of claim 1, wherein the variant CrylJ polypeptide has at least 95% identity to SEQ ID NO:2. 4. The variant CrylJ polypeptide of claim 1, wherein the variant CrylJ polypeptide has at least 95% identity to SEQ ID NO:4 or SEQ ID NO:8. 5. The variant Cry1J polypeptide according to claim 1, 2, 3 or 4, wherein said variant Cry1J polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:8. 6. A recombinant nucleic acid molecule encoding the variant Cry1J polypeptide according to claim 1, 2, 3, 4 or 5. 7. The recombinant nucleic acid molecule according to claim 6, wherein said nucleic acid sequence has been optimized for expression in plants. 8. The recombinant nucleic acid molecule according to claim 7, wherein said nucleic acid sequence is a synthetic nucleotide sequence having plant-preferred codons that has been designed for expression in plants. 9. The recombinant nucleic acid molecule according to claim 6, 7 or 8, wherein said nucleic acid sequence has been optimized for expression in soybean. 10. The recombinant nucleic acid molecule according to claim 6, wherein said nucleic acid is selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:7. 11. A DNA construct comprising a nucleic acid molecule according to any one of claims 6 to 10 and a heterologous regulatory element operably linked to said nucleic acid molecule. 12. A host cell comprising the DNA construct according to claim 11. 13. The host cell of claim 12, wherein the host cell is a bacterial cell. 14. The host cell of claim 12, wherein the host cell is a plant cell. 15. The host cell of claim 12, wherein the host cell is a soybean cell. 16. A transgenic plant comprising the host cell of claim 12. 17. The transgenic plant according to claim 16, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, cruciferous species, pepper species, potato, cotton, rice, soybean, sugar beet, sugar cane , tobacco, barley and canola. 18. A seed comprising the DNA construct of claim 11. 19. A composition comprising a variant Cry1J polypeptide according to any one of claims 1, 2, 3, 4 or 5. 20. The composition of claim 19, wherein the composition comprises 1% to 99% by weight of the variant Cry1J polypeptide. 21. A method of controlling a population of Lepidoptera pests, comprising contacting said population with a pesticidally effective amount of the variant Cry1J polypeptide according to any one of claims 1-5. 22. A method for killing Lepidoptera pests, comprising contacting said pests or feeding said pests with a pesticidally effective amount of the variant Cry1J polypeptide according to any one of claims 1-5. 23. A method for producing a polypeptide having pesticidal activity, comprising culturing the host cell according to any one of claims 12-15 under conditions in which the nucleic acid molecule encoding the variant Cry1J polypeptide is expressed. 24. A plant or plant cell having stably incorporated in its genome the DNA construct of claim 11. 25. A method of protecting a plant from insect pests, comprising introducing into said plant the DNA construct of claim 10. 26. The method of claim 24, wherein the insect pest is soybean armyworm (Chrysodeixis includesns).